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Presented  by 
Clifford  B.  Hunt,  D,  0, 


COLLEGE  OF  OSTEOPATHIC  PHYSICIANS 
AND  SURGEONS  •  LOS  ANGELES,  CALIFORNIA 


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UNIVERSJITY  OF  CALIFORNIA 

CALIFORNIA  COLLEGE  OF  MEDICINE 

IIRPft^Y 

JIIN     81971 

IRVINE,  CALIFORNIA  92664 


Digitized  by  tine  Internet  Archive 

in  2007  with  funding  from 

IVIicrosoft  Corporation 


http://www.arGhive.org/details/clinicaldiagOOtoddiala 


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Explanation  of  Plate  I 

Stained  with  Wright's  stain.  All  drawn  to  same  scale. 
I,  Normal  red  corpuscle  for  comparison;  2,  normoblasts,  one  with 
lobulated  nucleus;  3,  megaloblast  and  microblast.  The  megaloblast 
shows  a  considerable  degree  of  polychromatophilia;  4,  blood-plaques, 
one  lying  upon  a  red  corpuscle;  5,  lymphocytes,  large  and  small;  6, 
large  mononuclear  leukocyte;  7,  transitional  leukocyte;  8,  polymor- 
phonuclear neutrophilic  leukocytes;  9,  eosinophilic  leukocytes,  one 
ruptured;  10,  basophilic  leukocyte;  11,  neutrophilic  myelocyte.  The 
granules  are  sometimes  less  numerous  and  less  distinct  than  here  shown; 
12,  eosinophilic  myelocytes;  13,  basophilic  myelocyte;  14,  "irritation" 
or  "stimulation"  form,  with  small  vacuoles;  15,  degenerated  leukocytes: 
two  polymorphonuclear  neutrophiles,  one  ruptured,  one  swollen  and 
vacuolated;  and  a  "basket  cell"  composed  of  an  irregular  meshwork 
of  nuclear  material;  16,  large  mononuclear  leukocyte  containing  pigment- 
granules;  from  a  case  of  tertian  malaria;  17,  four  stages  in  the  asexual 
cycle  of  the  tertian  malarial  parasite:  the  second  and  fourth  were  drawn 
from  tlie  same  slide  taken  from  a  case  of  double  tertian;  18,  red  corpuscle 
containing  tertian  parasite  and  showing  malarial  stippling;  ig,  estivo- 
autumnal  malarial  parasites:  two  small  ring  forms  within  the  same 
red  cell,  and  a  crescent  with  remains  of  the  red  corpuscle  in  its  concavity. 


Clinical  Diagnosis 

A  MANUAL  OF  LABORATORY  METHODS 


BY 

JAMES    CAMPBELL    TODD.    Ph.  B..   M.  D. 

PROFESSOR  OF  PATHOLOGY,  UNIVERSITY  OP  COLORADO 


Illustrated 


Third  Edition,  Revised  and  Enlarged 


PHILADELPHIA   AND  LONDON 

W.     B.     SAUNDERS     COMPANY 

I9I4 


QY4 


Copyright,  1908,  by  W.  B.  Saunders  Company.      Revised,  reprinted,  and 

recopyrighted  January,   J913.     Reprinted  July,   1913.     Revised, 

entirely  reset,  reprinted,  and  recopyrighted  October,  1914 


Copyright,  1914,  by  W.  B.  Saunders  Company 


PRINTED    IN    AMERICA 

PRESS   Of 
B.     SAUNDERS     COMPANV 
PHILADELPHIA 


TO 

MY  FATHER 

THESE    PAGES  ARE 
AFFECTIONATELY    DEDICATED 


7 

v.  3 


PREFACE  TO  THE  THIRD  EDITION 

:^  

'    In  the  present  edition,  as  in  the  preceding  one,  the 

;^  scope  of  this  book  has  been  somewhat  enlarged  and  its 

size  increased.     It  is  beheved  that  its  value  has  thereby 

^^  been  enhanced  without  sacrifice  of  the  simplicity  and 

^conciseness  which  were  its   original   aim.     As   before, 

chief  emphasis    has  been   placed    upon  methods  and 

^  microscopic  morphology  rather  than  upon  the  clinical 

,  significance  of  findings. 

Much  of  the  new  material  is  the  outgrowth  of  ques- 
tions which  have  arisen  in  class  and  laboratory.      To 
one  who  sees  a  great  deal  of  the  work  of  students  in 
the  clinical  laboratory  it  soon  becomes  evident  that 
^     errors  in  microscopic  diagnosis  spring  much  less  fre- 
^^quently  from  ignorance  of  the  typical  appearance  of 
C'microscopic  structures  than  from  imperfect  preparation 
of  the  slide,  faulty  manipulation  of  the  microscope,  or 
failure  to  recognize  extraneous  matters,  artefacts,  and 
other  misleading  appearances.     Such  sources  of  error 
have  been  given  especial  attention. 

Each   section   has  been   carefully  revised   and   con- 

"siderable  additions  have  been  made  to  every  chapter, 

{notably  to  those  on  Sputum,  Urine,  and  Animal  Para- 

~  sites.     The  chapter  upon  the  Use  of  the  Microscope  has 

been  enlarged  by  the  addition  of  many  practical  points. 


8  PREFACE 

including  a  set  of  practical  exercises  which  it  is  hoped 
will  prove  useful  to  the  student.  The  section  upon 
Cerebrospinal  Fluid  has  been  rewritten  in  the  light  of 
recent  advances  in  knowledge  of  this  fluid.  Among  the 
newer  methods  which  appear  in  this  edition  may  be  men- 
tioned :  the  newly  simplified  Volhard  method  for  chlorids 
in  urine;  the  urease  methods  for  urea  in  urine,  blood,  and 
spinal  fluid;  the  Rimini-Burnam  test  for  formaldehyd 
in  urine;  the  Weisz  permanganate  (urochromogen)  test; 
the  use  of  edestin  as  a  control  for  the  glycyl-tryptophan 
test  for  gastric  cancer;  Hun  toon's  method  for  spores; 
Ponder's  stain  for  diphtheria  bacilli;  and  the  luetin  re- 
action. 

A  new  chapter  on  Serodiagnostic  Methods,  including 
Abderhalden's  test  for  pregnancy,  the  complement  fixa- 
tion tests  for  syphilis,  gonorrhea,  tuberculosis,  and  can- 
cer, and  the  cobra-venom  test  for  syphilis,  has  been  added. 
This  is  from  the  pen  of  Ross  C.  Whitman,  B.  A.,  M.  D., 
Professor  of  Surgical  Pathology  and  Serology  in  the  Uni- 
versity of  Colorado. 

Thirty-five  new  pictures  have  been  included.  Some  of 
these  replace  poorer  pictures  of  the  last  edition,  and  in 
some  cases  from  two  to  six  have  been  grouped  to  form  a 
single  illustration,  hence  the  total  number  of  figures  in 
the  book  has  not  been  greatly  increased.  The  majority 
of  the  new  pictures  are  photomicrographs.  Inadequate 
as  is  the  photomicrograph  in  some  fields,  its  superiority 
to  drawings  in  clinical  microscopy  can  hardly  be  ques- 
tioned. 

For  aid  of  various  sorts  in  the  preparation  of  this  edi- 
tion the  author  wishes  to  acknowledge  his  indebtedness 
to  the  following  members  of  the  faculty  of  the  University 


PREFACE  9 

of  Colorado:  Clough  T.  Burnett,  M.  D.,  Professor  of 
Bacteriology;  Alvin  R.  Peebles,  M.  D.,  Professor  of 
Preventive  and  Experimental  Medicine;  Max  M.  Ellis, 
Ph.D.,  Assistant  Professor  of  Biology;  and  Charles  F. 
Poe,  M.  A.,  Ph.  C,  Instructor  in  Chemistry. 

J.  C.  T. 

Boulder,  Colorado, 

October,  1^14. 


PREFACE 


This  book  aims  to  present  a  clear  and  concise  state- 
ment of  the  more  important  laboratory  methods  which 
have  clinical  value,  and  a  brief  guide  to  interpretation 
of  results.  It  is  designed  for  the  student  and  practi- 
tioner, not  for  the  trained  laboratory  worker.  It  had 
its  origin  some  years  ago  in  a  short  set  of  notes  which 
the  author  dictated  to  his  classes,  and  has  gradually 
grown  by  the  addition  each  year  of  such  matter  as  the 
year's  teaching  suggested.  The  eagerness  and  care  with 
which  the  students  and  some  practitioners  took  these 
notes  and  used  them  convinced  the  writer  of  the  need 
of  a  volume  of  this  scope. 

The  methods  offered  are  practical;  and  as  far  as 
possible  are  those  which  require  the  least  complicated 
apparatus  and  the  least  expenditure  of  time.  Simplicity 
has  been  considered  to  be  more  essential  than  absolute 
accuracy.  Although  in  many  places  the  reader  is  given 
the  choice  of  several  methods  to  the  same  end,  the 
author  believes  it  better  to  learn  one  method  well  than 
to  learn  several  only  partially. 

More  can  be  learned  from  a  good  picture  than  from 
any  description,  hence  especial  attention  has  been  given 
to  the  illustrations,  and  it  is  hoped  that  they  will  serve 
truly  to  illustrate.     Practically  all  the  microscopic  struc- 


13  PREFACE 

tures  mentioned,  all  apparatus  not  in  general  use,  and 
many   of  the  color  reactions  are  shown  in  the  pictures. 

Although  no  credit  is  given  in  the  text,  the  recent 
medical  periodicals  and  the  various  standard  works  have 
been  freely  consulted.  Among  authors  whose  writings 
have  been  especially  helpful  may  be  mentioned  v.  Jaksch, 
Boston,  Simon,  Wood,  Emerson,  Purdy,  Ogden,  Ewald, 
Ehrhch  and  Lazarus,  Da  Costa,  Cabot,  Osier,  Stengel, 
and  McFarland. 

The  author  wishes  hereby  to  express  his  indebtedness 
to  Dr.  J.  A.  Wilder,  Professor  of  Pathology  in  the  Den- 
ver and  Gross  College  of  Medicine,  for  aid  in  the  final 
revision  of  the  manuscript;  and  to  W.  D.  Engel,  Ph.D., 
Professor  of  Chemistry,  for  suggestions  in  regard  to  de- 
tection of  drugs  in  the  urine.  He  desires  to  acknowl- 
edge the  care  with  which  Mr.  Ira  D.  Cassidy  has  made 
the  original  drawings,  and  also  the  uniform  courtesy  of 
W.  B.  Saunders  Company  during  the  preparation  of 
the  book. 

J.  C.  T. 

Denver,  Colorado. 


CONTENTS 


INTRODUCTION  page 

Use  of  the  Microscope 17 

CHAPTER  I 

The  Sputam 48 

Physical  Examination 51 

Microscopic  Examination 53 

Unstained  Sputum 53 

Stained  Sputum 62 

Chemic  Examination 80 

Sputum  in  Disease 81 

CHAPTER  II 

The  Urine 85 

Physical  Examination 88 

Chemic  Examination 100 

Normal  Constituents 100 

Abnormal  Constituents 127 

Microscopic  Examination ; 173 

Unorganized  Sediments 175 

Organized  Sediments ■ 187 

Extraneous  Structures 208 

The  Urine  in  Disease 210 

CHAPTER  III 

The  Blood 218 

Coagulation 221 

Hemoglobin 224 

Enumeration  of  Erythrocytes 233 

Color  Index 244 

Volume  Index 245 

13 


14  CONTENTS 

PAGE 

Enumeration  of  Leukocytes 246 

Decrease  in  Number  of  Leukocytes 247 

Increase  in  Number  of  Leukocytes 247 

Leukocytosis 248 

Leukemia 253 

Method  of  Counting  Leukocytes 253 

Enimieration  of  Blood-plaques 258 

Study  of  Stained  Blood 261 

Making  and  Staining  Blood-films 261 

Study  of  Stained  Films 271 

Blood  Parasites 292 

Bacteria 292 

Animal  Parasites 295 

Tests  for  Recognition  of  Blood 306 

Special  Blood  Pathology 308 

Anemia 308 

Leukemia 314 

CHAPTER  IV 

The  Stomach 319 

Examination  of  the  Gastric  Contents 319 

Obtaining  the  Contents 320 

Physical  Examination 323 

Chemic  Examination 325 

Microscopic  Examination 339 

The  Gastric  Contents  in  Disease 342 

Additional  Examinations  which  Give  Information  as  to  the 

Condition  of  the  Stomach 344 

CHAPTER  V 

The  Feces 348 

Macroscopic  Examination 349 

Chemic  Examination 352 

Microscopic  Examination 355 

Functional  Tests 360 

CHAPTER  VI 

Animal  Parasites 363 

Protozoa 366 

Sarcodina. 368 

Mastigophora  (Flagellata) 372 


CONTENTS  15 

PAGE 

Spwrozoa 381 

Infusoria 382 

Platyhelminthes 383 

Nemathelminthes 402 

Arthropoda 417 

CHAPTER  VII 

MisceUaneous  Examinations 419 

Pus 419 

Peritoneal,  Pleural,  and  Pericardial  Fluids 423 

Cerebrospinal  Fluid 426 

Animal  Inoculation 432 

The  Mouth 433 

The  Eye 438 

The  Ear 440 

Parasitic  Diseases  of  the  Skin 441 

Milk 441 

Syphilitic  Material 445 

Semen 449 

Diagnosis  of  Rabies 451 

CHAPTER  VIII 

Bacteriologic  Methods 454 

Apparatus 454 

Sterilization 457 

Preparation  of  Culture-tubes 458 

Culture-media 459 

Staining  Methods 465 

Methods  of  Studying  Bacteria 471 

Characteristics  of  Special  Bacteria 475 

CHAPTER  IX 

Preparation  and  Use  of  Vaccines 479 

Preparation  of  Vaccine 479 

Method  of  Use 485 

Dosage 486 

Therapeutic  Indications 486 

Tuberculins 488 

Tuberculin  in  Diagnosis 49° 

Cutaneous  Test  for  Syphilis 49i 


l6  CONTENTS 

CIL\PTER  X  PAGE 

Serodiagnostic  Methods 493 

Immunity : 493 

Apparatus 49S 

Reactions  Based  Upon  Immune  Bodies  of  the  Second  Order. . .  497 

The  Widal  Reaction 497 

Biologic  Identification  of  Unknown  Proteins 503 

Opsonins 507 

Reactions  Based  Upon  Immune  Bodies  of  the  Third  Order 510 

Complement  Deviation  Test  for  Syphilis 511 

Complement  Deviation  Test  for  Gonorrhea 523 

Complement  Deviation  Test  for  Malignant  Disease 524 

Complement  Deviation  Test  for  Tuberculosis 526 

Cobra-venom  Test  for  Syphilis 528 

The  Protective  Ferment  Reactions 530 

The  Dialyzation  Method 532 

The  Polariscopic  Method 540 

APPENDIX 

Apparatus,  Reagents,  and  Stains 545 

Apparatus 545 

Reagents  and  Stains 547 

Weights,  Measures,  etc.,  with  Equivalents 553 

Temperature 554 


Index. 


555 


CLINICAL  DIAGNOSIS 


INTRODUCTION 
USE  OF  THE  MICROSCOPE 


There  is  probably  no 
laboratory  instrument 
whose  usefulness  de- 
pends so  much  upon 
proper  manipulation  as 
the  microscope,  and 
none  is  so  frequently 
misused  by  beginners. 
Some  suggestions  as  to 
its  proper  use  are,  there- 
fore, given  at  this  place. 
It  is  presumed  that  the 
reader  is  already  famil- 
iar with  its  general  con- 
struction (Fig.  i). 

For  those  who  wish 
to  understand  the  prin- 
ciples of  the  microscope 
and  its  manipulation — 
and  best  results  are  im- 
possible without  such  an 
understanding — a  care- 
ful study  of  some  stand- 

2 


Fig.  I. — Handle-arm  microscope:  E,  Eye- 
piece; D,  draw-tube;  T,  body-tube;  RN, 
revolving  nose-piece;  O,  objective;  PH,  pinion 
head;  MH,  micrometer  head;  HA,  handle-arm; 
SS,  substage;  S,  stage;  M,  mirror;  B,  base;  R, 
rack;  P,  pillar;  I,  inclination  joint. 

17 


l8  INTRODUCTION 

ard  work  upon  microscopy,  such  as  those  of  Carpenter, 
Spitta,  and  Sir  A.  E.  Wright,  is  earnestly  recommended. 
It  is  also  recommended  that  the  beginner  provide  him- 
self with  some  slides  of  diatoms,  for  example,  Pleuro- 
sigma  angulahim,  Surirella  gemma,  and  Amphipleura 
pellucida,  costing  fifty  cents  each,  and  with  some  good 
preparations  of  stained  and  unstained  blood.  The 
blood  slides  can  easily  be  made  from  one's  own  blood, 
as  described  in  Chapter  III.  Faithful  practice  upon 
such  test-objects,  in  the  light  of  the  principles  of  micros- 
copy, will  enable  the  student  to  reach,  intelligently,  an 
accuracy  in  manipulation  to  which  the  ordinary  labora- 
tory worker  attains  only  slowly  and  by  rule  of  thumb. 
He  will  soon  find  that  the  bringing  of  an  object  into  ac- 
curate focus  is  by  no  means  all  of  microscopy. 

Illumination. — Good  work  cannot  be  done  without 
proper  illumination.  It  is  difficult  to  lay  too  much 
stress  upon  this  point. 

The  Ught  which  is  generally  recommended  as  best  is 
that  from  a  white  cloud,  the  microscope  being  placed 
by  preference  at  a  north  window,  to  avoid  direct  sun- 
Hght.  At  any  other  window  a  white  window-shade  is 
desirable.  Such  light  is  satisfactory  for  all  ordinary  work. 
Artificial  light  is,  however,  imperative  for  those  who 
must  work  at  night,  and  is  a  great  convenience  at  all 
times.  Properly  regulated  artificial  Hght,  moreover, 
offers  decided  advantages  over  daylight  for  critical  work. 
Almost  any  strong  light  which  is  diffused  through  a 
frosted  globe  will  give  fair  results.  The  inverted  Wels- 
bach  light  with  such  a  globe  is  excellent.  The  follow- 
ing plan  is  much  used  abroad,  and  gives  results  equal 
to  the  best  daylight:  A  Welsbach  lamp  or  strong  elec- 


USE   OF   THE  MICROSCOPE 


19 


trie  light  is  used,  and  a  spheric  glass  globe — a  6-inch 
round-bottom  flask  answers  admirably — is  placed  be- 
tween it  and  the  microscope,  to  act  as  a  condenser  (Fig. 
2) .  The  flask  should  be  at  a  distance  equal  to  its  diameter 
from  both  the  light  and  the  mirror  of  the  microscope. 
In  order  to  filter  out  the  yellow  rays  the  flask  is  filled 
with  water  to  which  have  been  added  a  few  crystals 
of  copper  sulphate  and  a  little  ammonia. 


Fig.  2. — Illumination  with  water-bottle  condenser. 


For  critical  work,  the  method  suggested  by  Sir  A.  E. 
Wright  is  to  be  preferred.  He  has  shown  that  fog  is 
dispelled  and  definition  is  improved  if  the  size  of  the 
light  source  is  so  regulated  that  its  image,  thrown  upon 
the  slide  by  the  condenser,  coincides  with  the  real  field 
of  the  objective.  Upon  this  principle  a  very  neat  and 
satisfactory  microscope  lamp,  shown  in  Fig.  3,  has  been 
designed  by  B.  H.  Matthews.  It  is  fitted  with  iris- 
diaphragm,   condensing  lens,   small  electric  light,   and 


20 


INTRODUCTION 


reflector,  and  has  a  slot  in  which  a  ray  filter  or  ground- 
glass  disk  may  be  inserted. 

Illumination  may  be  either  central  or  obliqtte,  depend- 
ing upon  the  direction  in  which  the  light  enters  the 
microscope.  To  obtain  central  illumination,  the  mirror 
should  be  so  adjusted  that  the  light  from  the  source 
selected  is  reflected  directly  up  the  tube  of  the  micro- 
scope.    This  is  easily  done  by  removing  the  eye-piece 


Fig.  3. — Matthews'  microscope  lamp  with  iris-diiiphragm. 


and  looking  down  the  tube  while  adjusting  the  mirror. 
The  eye-piece  is  then  replaced,  and  the  light  reduced  as 
much  as  desired  by  means  of  the  diaphragm. 

Oblique  illumination  is  obtained  in  the  more  simple 
instruments  by  swinging  the  mirror  to  one  side,  so  that 
the  light  enters  the  microscope  obliquely.  The  more 
complicated  instruments  obtain  it  by  means  of  a  rack 
and    pinion,    which    moves    the    diaphragm    laterally. 


USE   OF   THE   MICROSCOPE  21 

Beginners  frequently  use  oblique  illumination  without 
recognizing  it,  and  are  thereby  much  confused.  If  the 
light  be  obHque,  an  object  in  the  center  of  the  field  will 
appear  to  move  from  side  to  side  when  the  fine  adjust- 
ment is  turned  back  and  forth. 

The  amount  of  light  admitted  is  also  important.  It  is 
regulated  by  the  diaphragm. 

The  bulk  of  routine  work  is  done  with  central  illumi- 
nation, and,  therefore,  every  examination  should  begin 
with  it.  Each  of  the  forms  of  illmnination,  however — 
central  and  oblique,  subdued  and  strong — has  its  special 
uses  and  demands  some  consideration  here.  The  well- 
known  rule,  **Use  the  least  light  which  will  show  the 
object  well,"  is  good,  but  it  does  not  go  far  enough. 

In  studying  any  microscopic  structure  one  considers: 
(i)  its  color,  (2)  its  outline,  and  (3)  its  surface  contour. 
No  one  form  of  illumination  shows  aU  of  these  to  the 
best  advantage.  It  may,  therefore,  be  necessary  to 
change  the  illumination  many  times  during  a  micro- 
scopic examination.  To  see  color  best,  use  central  illu- 
mination with  strong  light.  The  principle  is  that  by 
which  a  stained  glass  window  shows  the  purest  color 
when  the  light  is  streaming  through  it.  Strong  central 
light  is,  therefore,  to  be  used  for  structures  such  as 
stained  bacteria,  whose  recognition  depends  chiefly  upon 
their  color,  and,  alternating  with  other  forms,  for  stained 
structures  in  general.  To  study  the  outline  of  an  object 
use  very  subdued  central  illumination.  The  diaphragm 
is  closed  to  the  point  which  trial  shows  to  be  best  in  each 
case.  This  illumination  is  required  by  delicate  colorless 
objects,  such  as  hyaline  tube-casts  and  cholesterin 
crystals,  which  are  recognized  chiefly  by  their  outline. 


22  INTRODUCTION 

The  usual  mistake  of  beginners  is  to  work  with  the  dia- 
phragm too  wide  open.  Strong  light  will  often  render 
semitransparent  structures  entirely  invisible  (Fig.  4). 
To  study  surface  contour  use  oblique  light  of  a  strength 
suited  to  the  color  or  opacity  of  the  object.  In  routine 
work  oblique  illumination  is  resorted  to  only  to  study 
more  fully  some  object  which  has  been  found  with  cen- 
tral illumination,  as,  for  instance,  to  demonstrate  the 
cylindric  shape  of  a  hyaline  tube-cast. 


a  b 

Fig.  4. — a.  Hyaline  casts,  one  containing  renal  cells;  properly  subdued  illumination; 
h,  same  as  a;  strong  illumination.  The  casts  are  lost  in  the  glare,  and  only  the  renal  cells 
are  seen.     (From  Greene's  "Medical  Diagnosis"). 

Dark  Ground  Illumination. — This  consists  in  blocking 
out  the  central  rays  of  light  and  directing  the  peripheral 
rays  against  the  microscopic  object  from  the  side.  Only 
those  rays  which  strike  the  object  and  are  reflected 
upward  pass  into  the  objective.  The  object  thus  appears 
bright  upon  a  black  background.  By  means  of  this  form 
of  illumination  very  minute  structures  can  be  seen,  just 
as  particles  of  dust  in  the  atmosphere  become  visible 
when  a  ray  of  sunlight  enters  a  darkened  room. 

Dark  ground  illumination  for  low-power  work  can  be 


USE    OF   THE   MICROSCOPE  23 

obtained  by  means  of  the  ring  stops  with  central  disks 
which  accompany  most  microscopes  when  purchased. 
The  stop  is  placed  in  a  special  ring  beneath  the  con- 
denser. When  the  regular  stop  is  not  at  hand,  one  can 
use  an  extra  large  round  cover-glass,  in  the  center  of 
which  is  pasted  a  circular  disk  of  black  paper.  The  size 
of  the  black  disk  depends  upon  the  aperture  of  the  ob- 
jective with  which  it  is  to  be  used,  and  can  be  ascertained 
by  trial. 

For  oil-immersion  work  a  special  condenser  is  neces- 
sary. This  is  sold  under  the  name  of  reflecting  condenser, 
"dunkelfeld,"  dark  field  illuminator,  etc.  With  some 
makes  it  is  placed  upon  the  stage  of  the  microscope; 
with  others  it  is  substituted  for  the  regular  condens'.r. 
It  requires  an  intense  light,  like  direct  sunlight  or  a  snail 
arc-Hght. 

The  chief  use  of  dark  ground  illumination  in  clinical 
work  is  for  demonstration  of  Treponema  pallidum  in 
fresh  material  (see  Fig.  166). 

The  Condenser. — For  the  work  of  the  clinical  labora- 
tory a  substage  condenser  is  a  necessity.  Its  purpose 
is  to  condense  the  light  upon  the  object  to  be  examined. 
For  critical  work  the  light  must  be  focused  on  the  object 
by  raising  or  lowering  the  condenser  by  means  of  the 
screw  provided  for  the  purpose.  The  image  of  the  light 
source  will  then  appear  in  the  plane  of  the  object.  This 
is  best  seen  by  using  a  low-power  objective  and  ocular. 
Should  the  image  of  the  window-frame  or  other  nearby 
object  appear  in  the  field  and  prove  annoying,  the  con- 
denser may  be  raised  or  lowered  a  little.  It  is  often 
advised  to  remove  the  condenser  for  certain  kinds  of 


.24  INTRODUCTION 

work,  but  this  is  not  necessary  and  is  seldom  desirable 
in  the  clinical  laboratory. 

The  condenser  is  constructed  for  parallel  rays  of  light. 
With  daylight,  therefore,  the  plane  mirror  should  be 
used;  while  for  the  divergent  rays  of  ordinary  artificial 
light  the  concave  mirror,  which  tends  to  bring  the  rays 
together,  is  best. 

It  is  very  important  that  the  condenser  be  accurately 
centered  in  the  optical  axis  of  the  instrument,  and  most 
high-grade  instruments  have  centering  screws  by  which 
it  can  be  adjusted  at  any  time.  The  simplest  way  to 
recognize  whether  the  condenser  is  centered  is  to  close 
the  diaphragm  beneath  it  to  as  small  an  opening  as 
possible,  then  remove  the  eye-piece  and  look  down  the 
tube.  If  the  diaphragm  opening  does  not  appear  in  the 
center  of  the  field,  the  condenser  is  out  of  center. 

The  use  of  the  condenser  is  further  discussed  in  the 
following  sections. 

Objectives  and  Eye-pieces. — Unfortunately,  different 
makers  use  different  systems  of  designating  their  lenses. 
The  best  system,  and  the  one  chiefly  used  in  this  country, 
is  to  designate  objectives  by  their  focal  lengths  in  milli- 
meters, and  eye-pieces  by  their  magnifying  power, 
indicated  by  an  "  X."  Most  foreign  makers  use  this 
system  for  their  high-grade  lenses,  but  stiU  cling  to 
arbitrary  letters  or  numbers  for  their  ordinary  output. 

Objectives  are  of  two  classes — achromatic  and  apo- 
chromatic.  Those  in  general  use  are  of  the  achromatic 
type,  and  they  fulfil  all  requirements  for  ordinary  work. 
Apochromatic  objectives  are  more  highly  corrected  for 
chromatic  and  spheric  aberration,  and  represent  the 
highest  type  of  microscope  lenses  produced.    They  are 


USE   OF   THE   MICROSCOPE  2$ 

very  desirable  for  photomicrographic  and  research  work, 
but  for  routine  laboratory  work  do  not  offer  advantages 
commensurate  with  their  great  cost.  They  require  the 
iise  of  special  "compensating"  eye-pieces. 

Objectives  are  "corrected"  for  use  under  certain 
fixed  conditions,  and  they  will  give  the  best  results  only 
when  used  under  the  conditions  for  which  corrected.  The 
most  important  corrections  are:  (a)  For  tube  length; 
(&)  for  thickness  of  cover-glass;  and  (c)  for  the  medium 
between  objective  and  cover-glass. 

{a)  The  tube  length  with  which  an  objective  is  to  be 
used  is  usually  engraved  upon  it — in  most  cases  it  is 
i6o  mm.  The  draw-tube  of  the  microscope  should  be 
pulled  out  until  the  proper  length  is  obtained,  as  indi- 
cated by  the  graduations  on  its  side.  When  a  nose-piece 
is  used,  it  adds  about  15  mm.  to  the  tube  length,  and 
the  draw-tube  must  be  pushed  in  for  that  distance. 

{h)  The  average  No.  2  cover-glass  is  about  the  thickness 
for  which  most  objectives  are  corrected — usually  0.17  or 
o.  18  mm.  One  can  get  about  the  right  thickness  by  buying 
No.  2  covers  and  discarding  the  thick  ones;  or  by  buying 
No.  I  covers  and  discarding  the  thinner  ones.  Slight  dif- 
ferences in  cover-glass  thickness  can  be  compensated  by 
increasing  the  length  of  tube  when  the  cover  is  too  thin, 
and  decreasing  it  when  the  cover  is  too  thick.  The  cor- 
rection necessary  will  depend  upon  the  focal  length  and 
numeric  aperture  of  the  objective.  With  a  4-mm.  ob- 
jective of  0.85  numeric  aperture  a  difference  of  0.03  mm. 
in  cover-glass  thickness  requires  a  change  of  30  mm. 
in  the  tube  length.  Many  high-grade  objectives  are 
supplied  with  a  "correction  collar,"  which  accomplishes 
the  same  end.     While  for  critical  work,  especially  with 


26  INTRODUCTION 

apochromatics,  cover-glass  thickness  is  very  important, 
one  pays  little  attention  to  it  in  the  clinical  laboratory. 
A  high-power  dry  lens  always  requires  a  cover,  but  its 
exact  thickness  is  unimportant  in  routine  work.  Very 
low-power  and  oil-immersion  objectives  may  be  used 
without  any  cover-glass. 

(c)  The  correction  for  the  medium  between  objective 
and  cover-glass  is  very  important.  This  medium  may  be 
either  air  or  some  fluid,  and  the  objective  is  hence  either 
a  "dry"  or  an  "immersion"  objective.  The  immersion 
fluid  generally  used  is  cedar  oil,  which  gives  great  optical 
advantages  because  its  index  of  refraction  is  the  same  as 
that  of  crown  glass.  It  is  obvious  that  only  objectives 
with  very  short  working  distance,  as  the  2  mm.,  can  be 
used  with  an  immersion  fluid. 

To  use  an  oil-immersion  objective  a  drop  of  the  cedar 
oil  which  is  prepared  for  the  purpose  should  be  placed 
upon  the  cover,  and  the  objective  lowered  into  it.  A 
sUght  flash  of  Ught  will  be  seen  when  the  front  lens 
touches  the  oil.  The  objective  is  then  brought  to  a 
focus  in  the  usual  way.  In  order  to  avoid  bubbles  the 
oil  must  be  placed  upon  the  cover  carefully  and  without 
stirring  it  about.  Bubbles  are  a  frequent  source  of 
trouble,  and  should  always  be  looked  for  when  an  immer- 
sion objective  does  poor  work.  They  are  readily  seen  by 
removing  the  eye-piece  and  looking  down  the  tube. 
If  they  are  present,  the  oil  must  be  removed  and  a  new- 
drop  applied.  Immediately  after  use  both  objective 
and  slide  should  be  wiped  clean  with  lens-paper  or  a  soft 
linen  handkerchief.  In  an  emergency  glycerin  may  be 
used  instead  of  cedar  oil,  but,  of  course,  with  inferior 
results. 


USE   OF   THE   MICROSCOPE  27 

Curvature  of  field,  through  which  it  is  impossible  to 
focus  both  center  and  periphery  sharply  at  the  same 
time,  is  a  very  noticeable  defect;  but  it  is  less  serious  than 
appears  at  first  sight,  particularly  for  visual  work.  It 
is  easily  compensated  by  frequent  use  of  the  fine  focusing 
adjustment.  Complete  flatness  of  field  cannot  be  at- 
tained without  sacrifice  of  other  and  more  desirable 
properties.  Some  of  the  finest  objectives  made,  notably 
the  apochromatics,  show  decided  curvature. 

The  working  distance  of  an  objective  should  not 
be  confused  with  its  focal  distance.  The  former  term 
refers  to  the  distance  between  the  front  lens  of  the  ob- 
jective, when  it  is  in  focus,  and  the  cover-glass.  It  is 
always  less  than  the  focal  distance,  since  the  ''focal 
point"  lies  somewhere  within  the  objective;  and  it 
varies  considerably  with  different  makes.  Long  working 
distance  is  a  very  desirable  feature.  Some  oil-immersion 
objectives  have  such  short  working  distance  that  only 
very  thin  cover-glasses  can  be  used. 

A  useful  pointer  can  be  made  by  placing  a  straight 
piece  of  a  hair  across  the  opening  of  the  diaphragm  of 
the  eye-piece,  cementing  one  end  with  a  tiny  drop  of 
balsam,  and  cutting  the  hair  in  two  in  the  middle  with 
small  scissors.  When  the  eye-piece  is  in  place,  the  hair 
appears  as  a  black  Hne  extending  from  the  periphery  to 
the  center  of  the  microscopic  field. 

The  formation  of  the  microscopic  image  demands 
brief  consideration.  The  rays  of  light  which  are  re- 
flected upward  from  the  mirror  and  which  pass  through 
the  object  are  brought  to  a  focus  in  a  magnified,  inverted 
real  image.  This  can  be  focused  to  appear  at  different 
levels,  but  when  the  microscope  is  used  in  the  ordinary 


28  INTRODUCTION 

way  it  is  formed  at  about  the  level  of  the  diaphragm 
in  the  ocular.  It  can  be  seen  by  removing  the  ocular, 
placing  a  piece  of  ground  glass  on  the  top  dt  the  tube, 
and  focusing  upon  it.  When  viewing  this  image  a  roll  of 
paper  or  a  cylindric  mailing  tube  should  be  used  to  ex- 
clude extraneous  hght.  This  image,  in  turn,  is  mag- 
nified by  the  eye-lens  of  the  ocular,  producing  a  second 
real  image,  which  is  again  inverted,  and,  therefore, 
shows  the  object  right  side  up.  This  can  be  seen  upon 
a  ground  glass  held  a  few  inches  above  the  ocular,  pro- 
vided strong  artificial  Hght  be  used  and  the  room  dark- 
ened. The  eye,  when  it  looks  into  the  microscope,  sees, 
not  this  real  image,  but  rather  an  inverted  virtual  image 
which  appears  about  250  mm.  (10  inches)  in  front  of  the 
eye. 

Numeric  Aperture. — This  expression,  usually  written 
N.  A.,  indicates  the  amount  of  hght  which  enters  an 
objective  from  a  point  in  the  microscopic  field.  In 
optical  language,  N.  A.  is  the  sine  of  one-half  the  angle  of 
aperture  multipUed  by  the  index  of  refraction  of  the 
medium  between  the  cover  and  the  front  lens.  Numeric 
aperture  is  extremely  important,  because  upon  it  de- 
pends resolving  power,  which  is  the  most  important 
projDerty  of  an  objective.^ 

Resolving  power  is  the  abiUty  to  separate  minute 
details  of  structure.  For  example,  the  dark  portions  of 
a  good  half-tone  picture  appear  gray  or  black  to  the  un- 
aided eye,  but  a  lens  easily  resolves  this  apparently 

'  Resolving  power  really  depends  upon  two  factors,  the  N.  A.  and  the 
wave  length  of  light,  but  the  latter  can  be  ignored  in  practice.  The 
great  resolving  power  of  the  ultramicroscope  depends  upon  its  use  of 
light  of  short  wave  length. 


USE   OF   THE   MICROSCOPE  29 

uniform  surface  into  a  series  of  separate  dots.  Resolv- 
ing power  does  not  depend  upon  magnification.  The 
fine  lines  and  dots  upon  certain  diatoms  may  be  brought 
out  clearly  and  crisply  {i.  e.,  they  are  resolved)  by  an 
objective  of  high  numeric  aperture,  whereas  with  an  ob- 
jective of  lower  numeric  aperture,  but  greater  magnifying 
power,  the  same  diatom  may  appear  to  have  a  smooth 
surface,  with  no  markings  at  all,  no  matter  how  greatly 
it  is  magnified.  Knowing  the  N.  A.,  it  is  possible  to 
calculate  how  closely  Hues  and  dots  may  he  and  still 
be  resolved  by  a  given  objective.  To  state  the  numeric 
aperture,  therefore,  is  to  tell  what  the  objective  can 
accompHsh,  provided,  of  course,  that  spheric  and  chro- 
matic aberrations  are  satisfactorily  corrected.  An  ob- 
jective's N.  A.  is  usually  engraved  upon  the  mounting. 
It  is  an  important  fact,  and  one  almost  universally 
overlooked  by  practical  microscopists,  that  the  pro- 
portion of  the  numeric  aperture  of  an  objective  which  is 
utilized  depends  upon  the  aperture  of  the  cone  of  light 
delivered  by  the  condenser.  In  practice,  the  numeric 
aperture  of  an  objective  is  reduced  nearly  to  that  of 
the  condenser  (which  is  indicated  by  lower-case  letters, 
n.  a.).i  The  condenser  should,  therefore,  have  a  numeric 
aperture  at  least  equal  to  that  of  the  objective  with 
which  it  is  to  be  used.  Lowering  the  condenser  below 
its  focal  distance  and  closing  the  diaphragm  beneath 
it  have  the  effect  of  reducing  its  working  aperture.  A 
condenser,  whatever  its  numeric  aperture,  cannot 
deliver  through  the  air  a  cone  of  Hght  of  greater  n.  a. 

1  The  N.  A.  of  the  objective  is  not  reduced  wholly  to  that  of  the  con- 
denser, because,  owing  to  diffraction  phenomena,  a  small  part  of  the  un- 
illuminated  portion  of  the  black  lens  is  utilized. 


30  INTRODUCTION 

than  I.  From  these  considerations  it  follows  that  the 
proper  adjustment  of  the  substage  condenser  is  a  matter 
of  great  importance  when  using  objectives  of  high  N.  A., 
and  that,  to  gain  the  full  benefit  of  the  resolving  power 
of  such  objectives,  the  condenser  must  be  focused  on  the 
object  under  examination,  it  must  be  oiled  to  the  under 
surface  of  the  slide  in  the  same  way  as  the  immersion 
objective  is  oiled  to  the  cover-glass,  and  the  substage 
diaphragm  must  be  wide  open.  The  last  condition  in- 
troduces a  difficulty  in  that  colorless  structures  will  ap- 
pear "fogged"  in  a  glare  of  light  (see  Fig.  4) .  Wright  sug- 
gests that  the  size  of  the  Ught  source  be  so  regulated  by 
a  diaphragm  that  its  image,  thrown  on  the  slide  by  the 
condenser,  coincides  with  the  real  field  of  the  objective, 
and  maintains  that  in  this  way  it  is  possible  to  reduce 
the  glare  of  Hght  and  to  dispel  the  fog  without  closing 
the  diaphragm  of  the  condenser. 

One  can  easily  determine  how  much  of  the  aperture 
of  an  objective  is  in  use  by  removing  the  eye-piece,  look- 
ing down  the  tube,  and  observing  what  proportion  of  the 
back  lens  of  the  objective  is  illuminated.  The  relation 
of  the  illuminated  central  portion  to  the  unilluminated 
peripheral  zone  indicates  the  proportion  of  the  numeric 
aperture  in  use.  The  effect  of  raising  and  lowering  the 
condenser  and  of  oiling  it  to  the  slide  can  thus  be  easily 
seen. 

Another  property  of  an  objective  which  depends 
largely  upon  N.  A.  is  depth  of  focus,  the  ability  to  render 
details  in  different  planes  clearly  at  the  same  time.  The 
higher  the  N.  A.  and  the  greater  the  magnification,  the 
less  the  depth  of  focus.  Any  two  objectives  of  the  same 
focal  length  and  same  N.  A.  will  have  exactly  the  same 


USE   OF   THE   MICROSCOPE  3 1 

depth  of  focus.  Depth  of  focus  can  be  increased  by 
closing  down  the  diaphragm,  and  thus  reducing  the  N. 
A.  Great  depth  is  desirable  for  certain  low-power  work, 
but  for  high  powers  it  does  not  offer  advantages  to  bal- 
ance the  loss  of  N.  A.  by  which  it  is  attained.  In  some 
cases,  indeed,  it  is  a  real  disadvantage. 

Magnification. — The  degree  of  magnification  should 
always  be  expressed  in  diameters,  not  times,  which  is  a 
misleading  term.  The  former  refers  to  increase  of 
diameter;  the  latter,  to  increase  of  area.  The  compara- 
tively low  magnification  of  loo  diameters  is  the  same  as 
the  apparently  enormous  magnification  of  10,000  times. 

The  magnifying  power  of  a  lens  is  ascertained  by 
dividing  250  mm.,  or  10  inches  (the  distance  of  normal 
vision),  by  the  focal  length  of  the  lens.  The  focal  length 
of  an  objective  is  approximately  twice  the  diameter  of 
the  front  lens.  Thus,  the  2-mm.  objective  gives  a  mag- 
nification of  125  diameters;  the  25-mm.  eye-piece  gives 
a  magnification  of  lo  diameters,  and  is  usually  designated 
as  a  10  X  eye-piece.  When  an  objective  and  eye-piece 
are  used  together,  the  total  magnification  is  the  product 
of  the  two.  In  the  case  just  cited  the  total  magnifica- 
tion would  be  1250  diameters. 

It  is  easy  to  find  the  magnifying  power  of  any  combina- 
tion of  objective  and  ocular  by  actual  trial.  Place  the  count- 
ing slide  of  the  hemacytometer  upon  the  microscope  and  focus 
the  ruled  lines.  Now  adjust  a  sheet  of  paper  upon  the  table 
close  to  the  microscope  in  such  a  position  that  when  the  left  eye 
is  in  its  proper  place  at  the  ocular  the  paper  will  lie  in  front 
of  the  right  eye  at  the  normal  visual  distance,  i.  e.,  250  mm. 
(10  inches).  (The  paper  may  be  supported  upon  a  book,  if 
necessary.)    If  both  eyes  are  kept  open,  the  ruled  lines  will 


32  INTRODUCTION 

appear  to  be  projected  on  the  paper.  With  a  pencil  mark 
their  apparent  location  on  the  paper,  and  measure  the  dis- 
tance between  the  marks.  Divide  this  distance  by  0.05  mm., 
which  is  the  actual  distance  between  the  lines  on  the  slide. 
The  quotient  gives  the  magnification.  If,  to  take  an  example, 
the  lines  in  the  image  on  the  paper  are  5  najn.  apart,  the  mag- 
nification is  100  diameters.  The  figures  obtained  in  this  way 
will  vary  somewhat  as  one  is  near  or  far  sighted,  unless  the 
defect  of  vision  is  corrected  with  glasses. 

In  practice,  magnification  can  be  increased  in  one  of 
three  ways: 

(a)  Drawing  Out  the  Tube. — Since  the  increased  tube 
length  interferes  with  spheric  correction,  it  should  be 
used  only  with  the  knowledge  that  an  imperfect  image 
will  result.  ^ 

(b)  Using  a  Higher  Power  Objective. — As  a  rule,  this 
is  the  best  way,  because  resolving  power  is  also  in- 
creased; but  it  is  often  undesirable  because  of  the  shorter 
working  distance,  and  because  the  higher  objective  often 
gives  greater  magnification  than  is  desired,  or  cuts  down 
the  size  of  the  real  field  to  too  great  an  extent. 

(c)  Using  a  Shorter  Eye-piece. — This  is  the  simplest 
method.  It  has,  however,  certain  limitations.  When 
too  high  an  eye-piece  is  used,  there  results  a  hazy  image 
in  which  no  structural  detail  is  seen  clearly.  This  is 
called  "empty  magnification,"  and  depends  upon  the 
fact  that  the  objective  has  not  sufficient  resolving  power 
to  support  the  high  magnification.  It  has  been  aptly 
compared  to  the  enlargement,  by  stretching  in  all  direc- 
tions, of  a  picture  drawn  upon  a  sheet  of  rubber.  No 
new  detail  is  added,  no  matter  how  great  the  enlarge- 
ment.   The  extent  to  which  magnification  can  be  satis- 


USE   OF   THE   MICROSCOPE  33 

factorily  increased  by  eye-piecing  depends  wholly  upon 
the  resolving  power  of  the  objective,  and  consequently 
upon  the  N.  A.  The  greatest  total  or  combined  magni- 
fication which  will  give  an  absolutely  crisp  picture  is 
found  by  multiplying  the  N.  A.  of  an  objective  by  400. 
The  greatest  magnification  which  can  be  used  at  all 
satisfactorily  is  1000  times  the  N.  A.  For  example: 
The  ordinary  2-mm.  objective  has  a  N.  A.  of  1,30;  the 
greatest  magnification  which  will  give  an  absolutely 
sharp  picture  is  520  diameters,  which  is  obtained  ap- 
proximately by  using  a  4X  eye-piece.  Higher  eye- 
pieces can  be  used,  up  to  a  total  magnification  of  1300 
diameters  (10 X  eye-piece),  beyond  which  the  image 
becomes  wholly  unsatisfactory. 

The  Microscope  in  Use. — Optically,  it  is  a  matter  of 
indifference  whether  the  instrument  be  used  in  the 
vertical  position  or  inclined.  Examination  of  fluids  re- 
quires the  horizontal  stage,  and  since  much  of  the  work 
of  the  clinical  laboratory  is  of  this  nature  it  is  well  to 
accustom  one's  self  to  the  use  of  the  vertical  microscope. 

It  is  always  best  to  "focus  up,"  which  saves  annoy- 
ance and  probable  damage  to  slides  and  objectives. 
This  is  accomplished  by  bringing  the  objective  nearer 
the  slide  than  the  proper  focus,  and  then,  with  the  eye 
at  the  eye-piece,  turning  the  tube  up  until  the  object 
is  clearly  seen.  The  fine  adjustment  should  be  used  only 
to  get  an  exact  focus  with  the  higher  power  objectives  after 
the  instrument  is  in  approximate  focus.  It  should  not  be 
turned  more  than  one  revolution. 

There  will  be  less  fatigue  to  the  eyes  if  both  are  kept 
open  while  using  the  microscope,  and  if  no  effort  is  made 
to  see  objects  which  are  out  of  distinct  focus.  Fine 
3 


34  INTRODUCTION 

focusing  should  be  done  with  the  fine  adjustment,  not 
with  the  eye.  An  experienced  microscopist  keeps  his 
fingers  ahnost  constantly  upon  one  or  other  of  the  focus- 
ing adjustments. 

Although  the  ability  to  use  the  eyes  interchangeably 
is  sometimes  very  desirable,  greater  skill  in  recognizing 
objects  will  be  acquired  if  the  same  eye  be  always  used. 
The  left  eye  is  the  more  convenient,  because  the  right 
eye  is  thus  left  free  to  observe  the  drawing  one  may  wish 
to  do  with  the  right  hand.  After  a  little  practice  one 
can  cause  the  microscopic  image  to  appear  as  if  pro- 
jected upon  a  sheet  of  paper  placed  close  to  the  micro- 
scope under  the  free  eye.  This  gives  the  effect  of  a  cam- 
era lucida,  and  it  becomes  very  easy  to  trace  outlines. 
When  one  is  accustomed  to  spectacles,  they  should  not 
be  removed. 

To  be  seen  most  clearly,  an  object  should  be  brought 
to  the  center  of  the  field. 

One  often  wishes  to  mark  a  particular  field  upon  a 
permanent  preparation  so  as  to  refer  to  it  again.  The 
vernier  of  the  mechanical  stage  cannot  be  relied  upon, 
because  it  is  impossible  to  replace  the  stage  in  exactly 
the  same  position  after  it  has  been  removed  and  because 
its  position  is  frequently  changed  by  the  slight  knocks 
which  it  receives.  There  are  on  the  market  several 
"object  markers"  by  which  a  desired  field  can  be  marked 
with  ink,  or  by  a  circle  scratched  on  the  cover-glass  by 
a  minute  diamond,  while  the  slide  is  in  place  on  the  mi- 
croscope. The  circle  is  easily  located  with  a  low  power. 
In  the  absence  of  these,  one  can,  while  using  the  low 
power,  place  minute  spots  with  a  fine  pen  at  the  edge  of 
the  field  on  opposite  sides. 


USE   OF   THE  MICROSCOPE  35 

A  good  marking  material  is  a  cement  which  the  author 
has  long  used  for  making  cells,  ringing  cover-glasses,  etc. 
To  a  few  ounces  of  white  shellac  in  wood  alcohol  add  an  equal 
volume  of  gasoline,  shake  thoroughly,  and  let  stand  for 
twenty-four  hours,  or  until  well  separated  into  two  layers. 
Pipet  off  the  clear  lower  portion,  add  5  to  lo  drops  of  castor 
oil  to  each  ounce,  and  color  with  any  analin  dye  dissolved 
in  absolute  alcohol.  When  too  thick,  thin  with  absolute 
alcohol.  This  makes  a  beautiful,  transparent,  easy-flowing 
cement  which  does  not  crack  and  which  is  not  attacked  by 
xylol.  Glycerin  mounts  which  the  writer  ringed  with  it 
twenty  years  ago  are  still  in  perfect  condition. 

Many  good  workers  advise  against  the  use  of  spring 
clips  to  hold  the  slide  against  the  stage  of  the  microscope. 
Manipulation  of  the  slide  with  the  fingers  alone  certainly 
gives  good  training  in  delicacy  of  touch,  and  is  desirable 
when  examining  infectious  material  which  might  con- 
taminate the  clips,  but  is  most  annoying  in  practical 
work.  For  the  majority  of  examinations  it  is  much  more 
satisfactory  to  use  a  clip  at  one  end  of  the  slide,  with 
just  sufficient  pressure  to  hold  the  slide  without  inter- 
fering with  its  freedom  of  movement. 

Occasionally  when  one  wishes  a  very  low-power  ob- 
jective for  some  special  work  it  may  be  desirable  to 
unscrew  the  front  lens  of  the  i6-mm.  objective  and 
use  the  back  lens  only.  This  procedure  is  not  recom- 
mended for  critical  work,  and  it  should  not  be  tried 
with  high-power  objectives,  which  must  never  he  taken 
apart. 

To  attach  an  objective  it  should  be  supported  in 
position  against  the  nose-piece  by  means  of  the  index- 
linger  and  middle  finger,  which  grasp  it  as  one  would  a 


36  INTRODUCTION 

cigar.  It  is  then  screwed  into  place  with  the  fingers  of 
the  other  hand. 

Care  of  the  Microscope. — The  microscope  is  a  deli- 
cate instrument  and  should  be  handled  accordingly. 
Even  slight  disturbance  of  its  adjustments  may  cause 
serious  trouble.  It  is  so  heavy  that  one  is  apt  to  forget 
that  parts  of  it  are  fragile.  It  seems  uimecessary  to  say 
that  when  there  is  unusual  resistance  to  any  manipula- 
tion, force  should  never  be  used  to  overcome  it  until  its 
cause  has  first  been  sought;  and  yet  it  is  no  uncommon 
thing  to  see  students,  and  even  graduates,  push  a  high- 
power  objective  against  a  microscopic  preparation  with 
such  force  as  to  break  not  only  the  cover-glass,  but  even 
a  heavy  slide. 

It  is  most  convenient  to  carry  a  microscope  with  the 
fingers  grasping  the  pillar  and  the  arm  which  holds  the 
tube;  but  since  this  throws  a  strain  upon  the  fine  adjust- 
ment, it  is  safer  to  carry  it  by  the  base.  In  the  more 
recent  instruments  a  convenient  handle-arm  is  provided. 
To  bend  the  instrument  at  the  joint,  the  force  should 
be  applied  to  the  pillar  and  never  to  the  tube  or  the  stage. 

The  microscope  should  be  kept  scrupulously  clean, 
and  dust  must  not  be  allowed  to  settle  upon  it.  When 
not  in  use  the  instrument  should  be  kept  in  its  case  or 
under  a  cover.  An  expensive  glass  bell-jar  is  not  needed, 
and,  in  fact,  is  undesirable,  except  for  display.  It  is 
heavy  and  awkward  to  handle,  and  when  lifted  is  almost 
certain  (unless  great  care  is  exercised)  to  strike  the  mi- 
croscope. It  is  particularly  liable  to  strike  the  mechan- 
ical stage  and  disturb  its  adjustment.  The  simplest, 
cheapest,  lightest,  and  probably  the  best  cover  for  the 
microscope  is  a  truncated  cone  or  pyramid  of  pasteboard, 


USE   OF  THE  MICROSCOPE  37 

covered  with  creton  or  similar  material.  This  is  easily 
made  at  home. 

Lens  surfaces  which  have  been  exposed  to  dust  only 
should  be  cleaned  with  a  camel's-hair  brush.  A  small 
brush  and  a  booklet  of  lens-paper  should  always  be  at 
hand  in  the  microscope  case.  Those  surfaces  which  are 
exposed  to  finger-marks  should  be  cleaned  with  lens- 
paper,  or  a  soft  linen  handkerchief,  moistened  with  water 
if  necessary.  Particles  of  dirt  which  are  seen  in  the  field 
are  upon  the  sHde,  the  eye-piece,  or  the  condenser. 
Their  location  can  be  determined  by  moving  the  slide, 
rotating  the  eye-piece,  and  lowering  the  condenser. 
Dirt  upon  the  objective  cannot  be  seen  as  such;  it  causes 
a  diffuse  cloudiness.  When  the  image  is  hazy,  the  ob- 
jective probably  needs  cleaning;  or  in  case  of  an  oil- 
immersion  lens,  there  may  be  bubbles  in  the  oil. 

Oil  and  balsam  which  have  dried  upon  the  lenses  may 
be  removed  with  alcohol  or  xylol;  but  these  solvents 
must  be  used  sparingly  and  carefully,  as  there  is  danger 
of  softening  the  cement.  Some  manufacturers  now 
cement  their  lenses  with  shellac,  which  resists  xylol. 
Care  must  be  taken  not  to  get  any  alcohol  upon  the  brass 
parts,  as  it  will  remove  the  lacquer.  Balsam  and  dried 
oil  are  best  removed  from  the  brass  parts  with  xylol. 

When  the  vulcanite  stage  becomes  brown  and  dis- 
colored the  black  color  can  be  restored  by  rubbing  well 
with  petrolatum. 

Measurement  of  Microscopic  Objects. — Of  the  several 
methods,  the  most  convenient  and  accurate  is  the  use  of 
a  micrometer  eye-piece.  In  its  simplest  form  this  is 
similar  to  an  ordinary  eye-piece,  but  has  within  it  a  glass 
disk  upon  which  is  ruled  a  graduated  scale.    When  this 


38  INTRODUCTION 

eye-piece  is  placed  in  the  tube  of  the  microscope,  the 
ruled  lines  appear  in  the  microscopic  field,  and  the  size 
of  an  object  is  readily  determined  in  terms  of  the  divisions 
of  this  scale.  The  value  of  these  divisions  in  inches  or 
millimeters  manifestly  varies  with  different  magnifica- 
tions. Their  value  must,  therefore,  be  determined 
separately  for  each  objective.  This  is  accompUshed 
through  use  of  a  stage  micrometer— a  glass  slide  with 
carefully  ruled  scale  divided  into  hundredths  and  thou- 
sandths of  an  inch,  or  into  subdivisions  of  a  millimeter. 
The  stage  micrometer  is  placed  upon  the  stage  of  the 
microscope  and  brought  into  focus.  From  the  number 
of  divisions  of  the  eye-piece  scale  corresponding  to  each 
division  of  the  stage  micrometer  the  value  of  the  former 
in  fractions  of  an  inch  or  millimeter  is  easily  calculated. 
The  counting  slide  of  the  hemacytometer  wdll  answer 
in  place  of  a  stage  micrometer,  the  lines  which  form  the 
sides  of  the  small  squares  being  one-twentieth  of  a  milli- 
meter apart.  When  using  the  counting  chamber  with 
an  oil-immersion  lens  a  cover  must  be  used;  otherwise 
the  oil  will  fill  the  ruled  lines  and  cause  them  to  dis- 
appear. Any  eye-piece  can  be  converted  into  a  microm- 
eter eye-piece  by  placing  a  micrometer  disk — a  small 
circular  glass  plate  with  ruled  scale — ruled  side  down 
upon  its  diaphragm. 

The  following  method  of  micrometry  is  less  accurate, 
but  is  fairly  satisfactory  for  comparatively  coarse  objects, 
such  as  the  ova  of  parasites.  A  ruled  scale  corresponding 
to  the  magnified  image  of  the  hemacytometer  ruling  is 
drawn  upon  cardboard  in  the  manner  described  for  as- 
certaining magnifications  (see  p.  31).  This  card  may 
be  used  as  a  micrometer,  and  should  be  marked  with 


USE   OF   THE   MICROSCOPE  39 

the  value  of  its  graduations,  and  the  objective,  ocular, 
and  tube  length  with  which  it  is  to  be  used.  In  the 
example  cited  upon  p.  32  the  lines  are  5  mm.  apart,  cor- 
responding to  an  actual  distance  of  0.05  mm.  To  measure 
an  object,  the  cardboard  is  placed  in  the  position  which 
it  occupied  when  made  (at  the  right  of  the  microscope 
and  250  mm. — 10  inches — in  front  of  the  eye).  The  lines 
and  the  objects  on  the  shde  can  then  be  seen  together, 
and  the  space  covered  by  any  object  indicates  its  size. 


Fig.  5. — Egg  of  Taenia  saginata.     Lycopodium  granules  used  as  micrometer  (X  250) 
(photograph  by  the  author). 


The  graduations  made  as  above  indicated  are  too  coarse 
for  most  work,  and  they  should  be  subdivided.  If  five 
subdivisions  are  made,  each  will  have  a  value  of  lo  /U. 
Tuttle  has  suggested  that  in  feces  and  other  examina- 
tions a  little  lycopodium  powder  be  mixed  with  the 
material.  The  granules  are  of  uniform  size — 30  ^  in 
diameter — and  are  easily  recognized  (Fig.  5).  They 
furnish  a  useful  standard  with  which  the  size  of  other 
structures  can  be  compared.     Care  must  be  exercised 


40  INTRODUCTION 

not  to  use  too  much  powder.  The  lycopodium  is  con- 
veniently kept  in  a  gelatin  capsule,  and  a  faint  cloud  can 
be  dusted  over  the  slide  by  gently  scraping  the  edge  of 
the  lid  upon  the  rim  of  the  capsule. 

The  principal  microscopic  objects  which  are  measured 
clinically  are  animal  parasites  and  their  ova  and  abnor- 
mal blood-corpuscles.  The  metric  system  is  used  almost 
exclusively.  For  very  small  objects  o.ooi  mm.  has  been 
adopted  as  the  unit  of  measurement,  under  the  name 
micron.  It  is  represented  by  the  Greek  letter  [i.  For 
larger  objects,  where  exact  measurement  is  not  essential, 
the  diameter  of  a  red  blood-corpuscle  (7  to  8  |u)  is  some- 
times taken  as  a  unit. 

Photomicrography. — Although  high-grade  photomi- 
crography requires  expensive  apparatus  and  considerable 
skill  in  its  use,  fairly  good  pictures  of  microscopic  struc- 
tures can  be  made  by  any  one  with  simple  instruments. 

Any  camera  with  focusing  screen  or  a  Kodak  with 
plate  attachment  may  be  used.  It  is  best,  but  not  neces- 
sary, to  remove  the  photographic  lens.  The  camera  is 
placed  with  the  lens  (or  lens-opening,  if  the  lens  has  been 
removed)  looking  into  the  eye-piece  of  the  microscope, 
which  may  be  in  either  the  vertical  or  the  horizontal 
position.  One  can  easily  rig  up  a  standard  to  which  the- 
camera  can  be  attached  in  the  proper  position  by  means 
of  a  tripod  screw.  A  light-tight  connection  can  be  made 
of  a  cylinder  of  paper  or  a  cloth  sleeve  with  draw-strings. 
The  image  will  be  thrown  upon  the  ground-glass  focusing 
screen,  and  is  focused  by  means  of  the  fine  adjustment  of 
the  microscope.  The  degree  of  magnification  is  ascer- 
tained by  placing  the  ruled  slide  of  the  blood-counting 
instrument  upon   the  microscope   and  measuring   the 


USE   OF   THE   MICROSCOPE  41 

image  on  the  screen.  The  desired  magnification  is 
obtained  by  changing  objectives  or  eye-pieces  or  length- 
ening the  camera-draw. 

Focusing  is  comparatively  easy  with  low  powers,  but 
when  using  an  oil-immersion  objective  it  is  a  difficult 
problem  unless  the  source  of  light  be  very  brilliant.  If 
one  always  uses  the  same  length  of  camera  and  micro- 
scope tube,  a  good  plan  is  as  follows:  Ascertain  by  trial 
with  a  strong  light  how  far  the  fine  adjustment  screw 
must  be  turned  from  the  correct  eye  focus  to  bring  the 
image  into  sharp  focus  upon  the  ground-glass  screen. 
At  any  future  time  one  has  only  to  focus  accurately 
with  the  eye,  bring  the  camera  into  position,  and  turn 
the  fine  adjustment  the  required  distance  to  right  or 
left.  When  the  camera-draw  is  10  inches  no  change  in 
the  focusing  adjustment  will  be  necessary. 

The  Hght  should  be  as  intense  as  possible  in  order  to 
shorten  exposure,  but  any  light  that  is  satisfactory  for 
ordinary  microscopic  work  will  answer.  The  Hght  must 
be  carefully  centered.  It  is  nearly  always  necessary  to 
insert  a  color  screen  between  the  light  and  the  micro- 
scope. Pieces  of  colored  window-glass  are  useful  for 
this  purpose.  The  screen  should  have  a  color  comple- 
mentary to  that  which  it  is  desired  to  bring  out  strongly 
in  the  photograph:  for  blue  structures,  a  yellow  screen; 
for  red  structures,  a  green  screen.  For  the  average 
stained  preparation,  a  picric-acid  yellow  or  a  yellow 
green  will  be  found  satisfactory. 

Very  fair  pictures  can  be  made  on  Kodak  fihn,  but 
orthochromatic  plates  (of  which  Cramer's  "Iso"  and 
Seed's  "Ortho"  are  examples)  give  much  better  re- 
sults.    The  length  of  exposure  depends  upon  so  many 


42  INTRODUCTION 

factors  that  it  can  be  determined  only  by  trial.  It 
will  probably  vary  from  a  few  seconds  to  fifteen  minutes. 
Plates  are  developed  in  the  usual  way.  Either  the  tray 
or  tank  method  may  be  used,  but  in  order  to  secure  good 
contrast  it  is  often  desirable  to  overdevelop  somewhat. 
Metol-hydrochinon  is  an  excellent  developer,  as  it  gives 
good  contrast  with  full  detail. 

The  photograph  from  which  Fig.  6  was  made  was 
taken  with  a  Kodak  and  plate  attachment  on  an  "Iso" 


Fig.  6. — Leukemic  blood  (about  X  650).    rhoiograph  taken  with  a  Kodak,  as  described 
in  the  text. 

plate,  the  source  of  light  being  the  electric  lamp  and  con- 
densing lens  illustrated  in  Fig.  2.  It  was  focused  by  the 
method  described  above.  The  screen  was  a  picric-acid 
stained  photographic  plate.  Exposure,  three  and  a  half- 
minutes.  The  picture  loses  considerable  detail  in  repro- 
duction. 

Choice  of  a  Microscope. — It  is  poor  economy  to  buy 
a  cheap  instrument. 

For  the  work  of  a  clinical  laboratory  the  microscope 
should  preferably  be  of  the  new  handle-arm  type,  and 
should  have  a  large  stage.    It  should  be  provided  with  a 


USE   OF   THE    MICROSCOPE  43 

substage  condenser  (preferably  of  1.40  N.  A.),  three  or 
more  objectives  on  a  revolving  nose-piece,  and  two  or 
more  eye-pieces. 

The  most  generally  useful  objectives  are:  16  mm., 
4  mm.,  and  2  mm.  oil  immersion.  The  4-mm.  objective 
may  be  obtained  with  N.  A.  of  0.65  or  0.85.  If  it  is  to 
be  used  for  blood-counting,  the  former  is  preferable, 
since  its  working  distance  is  sufficient  to  take  the  thick 
cover  of  the  blood-counting  instrument.  For  coarse 
objects  a  32-mm.  objective  is  very  desirable.  The  eye- 
pieces most  frequently  used  are  4  X  and  8  X .  A  very  low 
power  (2X)  and  a  very  high  (18  X)  will  sometimes  be 
found  useful.  The  micrometer  eye-piece  is  almost  a 
necessity.  A  mechanical  stage,  preferably  of  the  attach- 
able type,  is  almost  indispensable  for  blood  and  certain 
other  work. 

A  first-class  microscope,  of  either  American  or  foreign 
make,  equipped  as  just  described,  will  cost  in  the  neigh- 
borhood of  $80  to  $100,  exclusive  of  the  mechanical  stage. 

Practical  Exercises. — The  following  is  a  brief  outfine 
of  certain  exercises  which  the  author  has  found  useful 
in  teaching  microscopy.  The  student  must  learn  as  early 
as  possible  what  can  be  expected  of  his  microscope  with 
proper  manipulation.  When  he  sits  down  to  work  his 
first  glance  should  tell  him  whether  the  instrument  is 
giving  its  best  results.  If  the  microscopic  picture  falls 
short  of  the  best,  he  must  locate  the  difiiculty  and  correct 
it  before  proceeding. 

.  I.  Clean  the  microscope  and  study  its  parts,  familiarizing 
yourself  with  the  names,  purposes,  and  movements  of  each 
(see  Fig.  i). 


44  INTRODUCTION 

2.  Practice  the  manipulations  necessary  to  locate  particles 
of  dust  or  dirt  which  appear  in  the  microscopic  field  (see  p.  37). 

3.  Try  the  effect  of  raising  and  lowering  the  condenser, 
and  of  changing  from  plane  to  concave  mirror,  upon  the 
image  of  nearby  objects  which  appear  in  the  microscopic 
field,  e.  g.,  the  window-frame  or  trees  outside  the  window. 
Note  that  such  objects  cause  an  unevenly  illuminated  or 
mottled  field  when  a  little  out  of  focus. 

4.  Insert  a  "pointer"  in  one  of  the  oculars  (see  p.  27). 

5.  Study  illumination.     Make  two  blood-films  on  slides. 

Stain  one  with  Wright's  or  Harlow's  stain.     Use 
one  unstained, 
(i)  Place  one  of  these  on  the  microscope,  bring  to  a 

focus,  and  practice  the  manipulations  necessary 

to  secure  (see  pp.  20,  21) — 

(a)  Central  illimiination. 

(b)  Oblique  illumination. 

(c)  Strong  and  subdued  illiunination. 

The  field  in  each  case  must  be  evenly  lighted  throughout, 
without  mottling.  Continue  until  you  can  adjust  any  de- 
sired form  of  illimiination  quickly  and  surely,  and  can  recog- 
nize each  by  a  glance  into  the  microscope, 

(2)  Using  the  stained  and  unstained  blood,  ascertain 

the  best  form  of  illumination  to  study  (see  pp. 

21,  22) — 
(a)  Outlines. 
{b)  Color. 

(c)  Surface  contour. 
Draw  the  corpuscles  as  they  appear  with  each. 

(3)  Try  dark  groimd  illumination  by  means  of  the  sub- 

stage  disk  (see  p.  23).  Study  the  imstained  blood- 
smear  and  draw  a  few  corpuscles.  Also  examine 
a  drop  of  saliva  covered  with  a  cover-glass.  Note 
the  epithelial  cells,  leukocytes,  and  miscellaneous 
bacteria.    Use  the  4-nun.  objective  for  this. 


USE  OF  THE  MICROSCOPE  45 

6.  With  central  illumination,  focus  upon  a  slide  and  ob- 
serve how  much  of  the  numeric  aperture  is  in  use  (see  p.  30). 
Try  the  effect  upon  numeric  aperture  of — 

(i)  Opening  and  closing  the  diaphragm. 

(2)  Raising  and  lowering  the  condenser. 

(3)  Using  the  oil-immersion  objective — 

(a)  Without  oil. 

(b)  With  oil  between  objective  and  cover-glass. 

(c)  With  oil  between  slide  and  condenser. 

7.  Upon  the  same  species  of  diatom  compare  two  object- 
ives of  3-mm.  focus  (therefore  of  same  magnifying  power), 
one  of  N.  A.  1.4  and  the  other  of  N.  A.  0.85.  They  will  be 
adjusted  by  the  instructor.  Note  the  superior  resolving 
power  of  the  lens  of  high  N.  A.  (see  p.  29), 

8.  Practice  using  the  oil-immersion  objective  (see  p.  26). 
If  there  is  difficulty  in  finding  the  specimen,  move  the  slide 
about  while  lowering  the  objective  to  a  focus.  Moving 
objects  will  catch  the  eye  as  the  ob  ective  approaches  the  cor- 
rect focus.  If  a  cover-glass  is  used,  its  edge  can  be  easily 
found  and  focused  upon. 

Produce  some  bubbles  in  the  oil  by  stirring  it  about  on 
the  slide;  observe  their  effect  on  the  microscopic  image,  and 
learn  to  detect  their  presence  (see  p.  26). 

9.  Image  formation  (see  p.  27).  Place  any  stained  prepa- 
ration upon  the  microscope.  Remove  the  ocular  and  use  a 
low-power  objective.  Lay  a  piece  of  ground  glass  across  the 
top  of  the  tube.  This  forms  a  screen  upon  which  an  image 
can  be  focused  by  means  of  the  coarse  adjustment.  Try 
focusing  it  at  different  levels.  Repeat  this  with  the  ocular 
in  place,  and  with  the  ground  glass  some  inches  above  the 
ocular.  These  exercises,  especially  the  last,  require  a  dark- 
ened room  and  strong  artificial  light. 

.  ID.  Find  by   trial   the  magnification  produced  by  your 
i6-mm.  objective  with  the  4X  ocular  (see  p.  31).    Compare 


46  INTRODUCTION 

your  result  with  that  obtained  by  calculation  from  the  focal 
length  (see  p.  31). 

11.  Micrometry. 

(i)  Evaluate  the  scale  of  your  micrometer  eye-piece 
with  a  high-power  objective,  and  measure  ac- 
curately 10  red  blood  corpuscles  and  10  leuko- 
cytes (see  pp.  37,  38). 

(2)  Prepare  a  cardboard  micrometer  and  measure  10 
lycopodium  granules  (see  pp.  38,  39). 

12.  Study  the  following  structures,  chiefly  with  a  view  to 
best  illumination.  Many  of  these  are  met  as  accidental  con- 
taminations in  microscopic  preparations  and  one  must  learn 
to  recognize  them.    Make  drawings  of  each. 

Fluids  are  examined  by  placing  a  drop  in  the  center  of  a 
clean  slide  and  applying  a  cover-glass.  The  drop  should  be 
large  enough  to  fill  the  space  between  the  slide  and  cover, 
but  not  large  enough  to  float  the  cover  about.  Fibers  or 
insoluble  powder  may  be  placed  in  a  drop  of  water  and 
covered. 

(i)  Air  bubbles  produced  by  shaking  a  little  diluted 
mucilage. 

(2)  Fresh  milk  diluted  with  three  or  four  volumes  of 

water.     Prepare  three  slides. 

(a)  Examine  one  untreated. 

(b)  Treat  one  with  solution  of  Sudan  III.     (For- 

method  see  p.  183.)  Note  color  assumed  by 
the  fat  globules.  This  is  one  of  the  most 
useful  tests  for  microscopic  fat. 

(c)  Treat  one  with  dilute  acetic  acid.    Note  clumping 

of  globules  similar  to  that  of  typhoid  bacilli  in 
the  Widal  test. 

(3)  A  drop  of  diluted  India-ink.      Note  the  dancing 

motion  of  the  particles  ("Brownian  motion"). 

(4)  Starch  granules.    Gently  scrape  the  freshly  cut  sur- 

face of  a  potato  with  a  knife,  place  the  scrapings 


USE   OF   THE   MICROSCOPE  47 

in  a  drop  of  water  on  a  slide,  remove  coarse 
particles,  and  apply  a  cover-glass.     Make  two 
preparations, 
(a)  Examine  one  untreated.     Note  the  variously 
sized  starch  granules,  oval,  colorless,  concen- 
trically striated. 
{b)  Treat  one  with  Lugol's  or  Gram's  iodin  solution. 
Note  change  in  color  of  granules.    This  is  the 
standard  test  for  starch. 

(5)  Yeast  which  has  been  growing  in  a  dextrose  solu- 

tion.    Make  two  preparations. 

(a)  Examine  one  unstained.    Note  "budding." 

(b)  Treat  one  with  iodin  solution.    Compare  color 

of  yeast  wuth  that  taken  by  starch. 

(6)  Mold  from  moldy  food.    Note  hyphae  and  spores. 

(7)  Various  fibers  and  other  structures  mounted  in  a 

drop  of  water. 

(c)  Cotton. 

(b)  Wool. 

(c)  Linen. 

(d)  Silk. 

(e)  Feather  tip. 

(/)  Some  dust  from  a  carpeted  room. 
(g)  A  hair  from  the  head. 

(8)  A  drop  of  decomposing  urine.     Note  bacteria  of 

various  kinds,  some  motile,  some  non-motile. 
Make  an  effort  to  distinguish  true  motility  from 
that  due  to  currents  in  the  fluid  and  to  "Brownian 
motion." 


CHAPTER  I 

THE  SPUTUM 

Preliminary  Considerations. — Before  beginning  the 
study  of  the  sputum,  the  student  will  do  well  to  familiar- 
ize himself  with  the  structures  which  may  be  present 
in  the  normal  mouth,  and  which  frequently  appear  in  the 
sputum  as  contaminations.  Nasal  mucus  and  material 
obtained  by  scraping  the  tongue  and  about  the  teeth 
should  be  studied  as  described  for  unstained  sputum.  A 
drop  of  Lugol's  solution  should  then  be  placed  at  the 
edge  of  the  cover,  and,  as  it  runs  under,  the  effect  upon 
different  structures  noted.  Another  portion  should  be 
spread  upon  slides  or  covers  and  stained  by  some  simple 
stain  and  by  Gram's  method.  The  structures  likely  to 
be  encountered  are  epithelial  cells  of  columnar  and 
squamous  types,  leukocytes,  food-particles,  Leptolhrix 
bticcalis,  and  great  numbers  of  saprophytic  bacteria, 
frequently  including  spirochetes.  These  structures  are 
described  later. 

When  collecting  the  sample  for  examination,  the 
morning  sputum,  or  the  whole  amount  for  twenty-four 
hours  should  be  saved.  In  beginning  tuberculosis 
tubercle  bacilli  can  often  be  found  in  that  first  coughed 
up  in  the  morning  when  they  cannot  be  detected  at  any 
other  time  of  day.  Sometimes,  in  these  early  cases, 
there  are  only  a  few  mucopurulent  flakes  which  contain 

48 


THE   SPUTUM  49 

the  bacilli,  or  only  a  small  purulent  mass  every  few  days, 
and  these  may  easily  be  overlooked  by  the  patient. 

Patients  should  be  instructed  to  rinse  the  mouth 
well  in  order  to  avoid  contamination  with  food-particles 
which  may  prove  confusing  in  the  examination,  and  to 
make  sure  that  the  sputum  comes  from  the  lungs  or 
bronchi  and  not  from  the  nose  and  nasopharynx.  Many 
persons  find  it  difficult  to  distinguish  between  the  two. 
It  is  desirable  that  the  material  be  raised  with  a  distinct 
expulsive  cough,  but  this  is  not  always  possible.  Ma- 
terial from  the  upper  air-passages  can  usually  be  iden- 
tified from  the  large  proportion  of  mucus  and  the  char- 
acter of  the  epithelial  cells. 

The  sputum  of  infants  and  young  children  is  usually 
swallowed  and  therefore  cannot  be  collected.  In  such 
cases  examination  of  the  feces  for  tubercle  bacilH  will 
sometimes  establish  a  diagnosis  of  tuberculosis. 

As  a  receptacle  for  the  sputum,  a  clean,  wide-mouthed 
bottle  with  tightly  fitting  cork  may  be  used.  The  pa- 
tient must  be  particularly  cautioned  against  smearing 
any  of  it  upon  the  outside  of  the  bottle.  This  is  prob- 
ably the  chief  source  of  danger  to  those  who  examine 
sputum.  Disinfectants  should  not  be  added.  Although 
some  of  them  (phenol,  for  example)  do  not  interfere 
with  detection  of  tubercle  bacilli,  they  generally  so  alter 
the  character  of  the  sputum  as  to  render  it  unfit  for 
satisfactory  examination. 

The  following  outline  is  suggested  for  the  routine 
examination : 

I.  Spread  the  material  in  a  thin  layer  in  a  Petri  dish  or 
between  two  plates  of  glass.    The  use  of  glass  plates  is  messy, 
4 


5©  THE   SPUTUM 

and  is  not  to  be  recommended  unless  the  top  plate  is  much 
smaller  than  the  lower  one,  or  has  some  sort  of  handle. 

2.  Examine  all  parts  carefully  with  the  naked  eye  (best 
over  a  black  background)  or  with  a  hand  lens.  The  portions 
most  suitable  for  further  examination  may  thus  be  easily 
selected.  This  macroscopic  examination  should  never  be 
omitted. 

3.  Transfer  various  portions,  including  all  suspicious 
particles,  to  clean  slides,  cover,  and  examine  unstained  with 
the  microscope  (see  p.  53). 

4.  Slip  the  covers  from  some  or  all  of  the  above  unstained 
preparations,  leaving  a  thin  smear  on  both  slide  and  cover. 

5.  Dry  and  fix  the  smears  and  stain  one  or  more  by  each 
of  the  following  methods: 

(c)  For  cells  and  bacteria  in  general,  using  pyronin- 
methyl  green  by  preference  (see  p.  467). 

(b)  For  tubercle  bacilli  (see  p.  63). 

(c)  Gram's  method  (see  p.  467). 

6.  When  indicated,  make  special  examinations  for — 

(a)  Capsules  of  bacteria  (see  p.  72). 

(b)  Eosinophilic  cells  (see  p.  77). 

(c)  Presence  of  albumin  (see  p.  80). 

After  the  examination  the  sputum  must  be  destroyed 
by  heat  or  chemicals,  and  everything  which  has  come  in 
contact  with  it  must  be  sterilized.  The  utmost  care 
must  be  taken  not  to  allow  any  of  it  to  dry  and  become 
disseminated  through  the  air.  If  files  are  about,  it  must 
be  kept  covered.  It  is  a  good  plan  to  conduct  the  exami- 
nation upon  a  large  newspaper,  which  can  then  be  burned. 
Contamination  of  the  work  table  is  thus  avoided.  If 
this  is  not  feasible,  the  table  should  be  washed  ofif  with  10 
per  cent,  lysol  or  other  disinfectant  solution,  and  allowed 
to  dry  slowly,  as  soon  as  the  sputum  work  is  finished. 


PHYSICAL   EXAMINATION  5 1 

Examination  of  the  sputum  is  most  conveniently  con- 
sidered under  four  heads:  I.  Physical  examination. 
II.  Microscopic  examination.  III.  Chemic  examination. 
IV.  Characteristics  of  the  sputum  in  various  diseases. 

I.  PHYSICAL  EXAMINATION 

1.  Quantity. — The  quantity  expectorated  in  twenty- 
four  hours  varies  greatly.  It  may  be  so  slight  as  to  be 
overlooked  entirely  in  beginning  tuberculosis.  It  is 
usually  small  in  acute  bronchitis  and  lobar  pneumonia. 
It  may  be  very  large — sometimes  as  much  as  looo  c.c. — 
in  advanced  tuberculosis  with  large  cavities,  edema  of  the 
lung,  bronchiectasis,  and  following  rupture  of  an  abscess 
or  empyema.  It  is  desirable  to  obtain  a  general  idea  of 
the  quantity,  but  accurate  measurement  is  unnecessary. 

2.  CoIor.^Since  the  sputum  ordinarily  consists  of 
varying  proportions  of  mucus  and  pus,  it  may  vary  from 
a  colorless,  translucent  mucus  to  an  opaque,  whitish  or 
yellow,  purulent  mass.  A  yellowish  green  is  frequently 
seen  in  advanced  phthisis  and  chronic  bronchitis.  In 
jaundice,  in  caseous  pneumonia,  and  in  slowly  resolv- 
ing lobar  pneumonia  it  may  assume  a  bright  green  color, 
due  to  bile  or  altered  blood-pigment. 

A  red  color  usually  indicates  the  presence  of  blood. 
Bright  red  blood,  most  commonly  in  streaks,  is  strongly 
suggestive  of  phthisis.  It  may  be  noted  early  in  the 
disease  and  generally  denotes  an  extension  of  the  tuber- 
culous process.  A  rusty  red  sputum  is  the  rule  in  croup- 
ous pneumonia,  and  was  at  one  time  considered  pathogno- 
monic of  the  disease.  'Trune-juice"  sputum  is  said  to 
be  characteristic  of  "drunkard's  pneumonia."  It  at 
least  indicates  a  dangerous  type  of  the  disease.    A  brown 


L     ll   I   <  I   ^ 


52  THE   SPUTUM 

color,  due  to  altered  blood-pigment,  follows  hemorrhages 
from  the  lungs,  and  is  present,  to  greater  or  less  degree, 
in  chronic  passive  congestion  of  the  lungs,  which  is  most 
'  frequently  due  to  a  heart  lesion. 

Gray  or  black  sputum  is  observed  among  those  who 
work  much  in  coal-dust,  and  is  occasionally  seen  in 
smokers  who  are  accustomed  to  "inhale." 

3.  Consistence. — ^According  to  their  consistence,  sputa 
are  usually  classified  as  serous,  mucoid,  purulent,  sero- 
purulent,  mucopurulent,  etc.,  which  names  explain 
themselves.  As  a  rule,  the  more  mucus  and  the  less  pus 
and  serum  a  sputimi  contains,  the  more  tenacious  it  is. 

The  rusty  sputum  of  croupous  pneumonia  is  extremely 
tenacious,  so  that  the  vessel  in  which  it  is  contained  may 
be  inverted  without  spilling  it.  The  same  is  true  of  the 
almost  purely  mucoid  sputum  ("sputum  crudum")  of 
beginning  acute  bronchitis,  and  of  that  which  follows 
an  attack  of  asthma.  A  purely  serous  sputum,  usually 
slightly  blood  tinged,  is  fairly  characteristic  of  edema  of 
the  lungs. 

4.  Dittrich's  Plugs. — While  these  bodies  sometimes 
appear  in  the  sputum,  they  are  more  frequently  expec- 
torated alone.  They  are  caseous  masses,  usually  about 
the  size  of  a  pin-head,  but  sometimes  reaching  that  of 
a  bean.  The  smaller  ones  are  yellow,  the  larger  ones 
gray.  When  crushed,  they  emit  a  foul  odor.  Micro- 
scopically, they  consist  of  granular  debris,  fat-globules, 
fatty  acid  crystals,  and  bacteria.  They  are  formed 
in  the  bronchi,  and  are  sometimes  expectorated  by 
healthy  persons,  but  are  more  frequent  in  putrid  bron- 
chitis and  bronchiectasis.  The  laity  commonly  regard 
them  as  evidence  of  tuberculosis.    The  similar  caseous 


'^f^YK^ 


MICROSCOPIC  EXAMINATION  53 

masses  which  are  formed  in  the  crypts  of  the  tonsils  are 
sometimes  also  included  under  this  name. 

5.  Bronchial  Casts. — Branching,  tree-like  casts  of  the 
larger  bronchial  tubes  are  sometimes  seen.  They  are 
best  examined  in  water.  Further  description  is  foimd 
on  p.  59. 

n.  MICROSCOPIC  EXAMINATION 

The  portions  most  likely  to  contain  structures  of 
interest  should  be  very  carefully  selected,  as  already 
described.  The  few  minutes  spent  in  this  preliminary 
examination  will  sometimes  save  hours  of  work  later. 
Opaque,  white  or  yellow  particles  are  most  frequently 
bits  of  food,  but  may  be  cheesy  masses  from  the  tonsils; 
small  cheesy  nodules,  derived  from  tuberculous  cavities 
and  containing  many  tubercle  bacilli  and  elastic  fibers; 
Curschmann's  spirals,  or  small  fibrinous  casts,  coiled  into 
little  balls;  or  shreds  of  mucus  with  great  numbers  of 
entangled  pus-corpuscles.  The  food-particles  most  apt 
to  cause  confusion  are  bits  of  bread,  which  can  be  recog- 
nized by  the  blue  color  which  they  assume  when  touched 
with  iodin  solution. 

Some  structures  are  best  identified  without  staining; 
others  require  that  the  sputum  be  stained. 

A.   Unstained  Sputum 

A  careful  study  of  the  unstained  sputum  should  be 
included  in  every  routine  examination.  Unfortunately 
it  is  almost  universally  neglected.  It  best  reveals  cer- 
tain structures  which  are  seen  imperfectly  or  not  at  all 
in  stained  preparations.  It  gives  a  general  idea  of  the 
other  structures  which  are  present,   such  as  pus-cor- 


54  THE   SPUTUM 

puscles,  eosinophiles,  epithelial  cells,  and  blood,  and 
thus  suggests  appropriate  stains  to  be  used  later.  It 
enables  one  to  select  more  intelligently  the  portions  to 
be  examined  for  tubercle  bacilli. 

The  particle  selected  for  examination  should  be  trans- 
ferred to  a  clean  slide,  covered  with  a  clean  cover-glass, 
and  examined  with  the  i6-mm.  objective,  followed  by 
the  4  mm.  It  is  convenient  to  handle  the  bits  of  sputum 
with  a  wooden  tooth-pick  or  with  a  wooden  cotton- 
applicator,  which  may  be  burned  when  done  with.  The 
platinum  wire  used  in  bacteriologic  work  is  unsatisfactory 
because  not  usually  stiff  enough.  A  little  practice  is 
necessary  before  one  can  handle  particles  of  sputum 
readily.  The  bit  desired  should  be  separated  from  the 
bulk  of  the  sputum  by  cutting  it  free  with  the  tooth- 
pick. It  can  then  be  picked  up  by  rotating  the  end  of  a 
fresh  tooth-pick  against  it.  The  slide  must  never  he 
dipped  into  the  sputum,  nor  must  any  of  the  sputum  he 
allowed  to  reach  its  edges  in  spreading. 

The  more  important  structures  to  be  seen  in  unstained 
sputum  are:  elastic  fibers,  Curschmann's  spirals,  Char- 
cot-Leyden  crystals,  fibrinous  casts,  the  ray  fungus  of 
actinomycosis,  and  molds.  Pigmented  cells,  especially 
the  so-called  ''heart-failure  cells"  (see  p.  79),  are  also 
best  studied  without  staining  (Plate  II,  Fig.  i). 

I.  Elastic  Fibers. — These  are  the  elastic  fibers  of 
the  pulmonary  substance,  where  they  are  distributed  in 
the  walls  of  the  alveoli,  the  bronchioles,  and  the  blood- 
vessels (Fig.  7) .  When  found  in  the  sputum  they  always 
indicate  destructive  disease  of  the  lung,  provided  they 
do  not  come  from  the  food,  which  is  a  not  infrequent 
source.     They  are  found  most  commonly  in  phthisis; 


MICROSCX)PIC   EXAMINATION 


55 


rarely  in  other  diseases.  Advanced  cases  of  tuberculosis 
often  show  great  numbers,  and,  rarely,  they  may  be 
found  in  early  tuberculosis  when  the  bacilli  cannot  be 
detected.  After  the  diagnosis  is  estabUshed  they  furnish 
a  valuable  clue  as  to  the  existence  and  rate  of  lung  de- 
struction. In  gangrene  of  the  lung,  contrary  to  the 
older  teaching,  elastic  tissue  is  probably  always  present 
in  the  sputum,  usually  in  large  fragments. 


Fig.  7. — Elastic  fibers  from  the  sputum:  a,  Highly  magnified;  b,  alveolar  arrangement, 
less  highly  magnified  (after  Bizzozero). 


The  fibers  should  be  searched  for  with  a  i6-inm.  ob- 
jective, although  a  higher  power  is  needed  to  identify 
them  with  certainty.  They  may  usually  be  more  clearly 
seen  if  a  drop  of  10  to  20  per  cent,  caustic  soda  solution 
be  mixed  with  the  sputum  on  the  slide  before  the  cover 
is  applied.  Under  the  4  mm.  they  appear  as  slender, 
highly  refractive  fibers  with  double  contour,  and  often 


56  THE   SPUTUM 

curled  or  split  ends.  Frequently  they  are  found  in  alve- 
olar arrangement,  retaining  the  original  outline  of  the 
alveoli  of  the  lung  (Fig.  7,  b).  This  arrangement  is 
positive  proof  of  their  origin  in  the  lung. 

Leptothrix  huccalis,  which  is  a  normal  inhabitant  of  the 
mouth,  may  easily  be  mistaken  for  elastic  tissue.  It 
can  be  distinguished  by  running  a  httle  iodin  solution 
under  the  cover-glass  (see  p.  73).  Fatty-acid  crystals, 
which  are  often  present  in  Dittrich's  plugs  and  in  sputum 
which  has  lain  in  the  body  for  some  time,  also  simulate 
elastic  tissue  when  very  long,  but  they  are  more  like  stiff, 
straight  or  curved  needles  than  wavy  threads.  They 
show  varicosities  when  the  cover-glass  is  pressed  upon. 
The  structures  which  most  frequently  confuse  the  stu- 
dent are  the  cotton  fibrils  which  are  present  as  a  contami- 
nation in  most  sputa.  These  are  usually  coarser  than 
elastic  fibers,  and  flat,  with  one  or  two  twists,  and  often 
have  longitudinal  striations  and  frayed-out  ends. 

To  find  elastic  fibers  when  not  abundant,  boil  the 
sputum  with  a  10  per  cent,  solution  of  caustic  soda  until 
it  becomes  fluid ;  add  several  times  its  bulk  of  water,  and 
centrifugalize,  or  allow  to  stand  for  twenty-four  hours  in 
a  conical  glass.  Examine  the  sediment  microscopically. 
The  fibers  will  be  pale  and  swollen  and,  therefore,  some- 
what difiicult  to  recognize.  Too  long  boiling  will  destroy 
them  entirely. 

The  above  procedure,  although  widely  recommended, 
will  rarely  or  never  be  necessary  if  the  sputmn  is  care- 
fully examined  in  a  thin  layer  against  a  black  back- 
ground macroscopically  and  with  a  hand-lens,  and  if  all 
suspicious  portions  are  further  studied  with  the  micro- 
scope. 


MICROSCOPIC   EXAMINATION 


57 


2.  Curschmann's  Spirals. — These  peculiar  structures 
are  found  most  frequently  in  bronchial  asthma,  of 
which  they  are  faily  characteristic.  Although  not  pres- 
ent in  every  attack,  they  probably  occur  at  some  time 
in  every  case.  Sometimes  they  can  be  foimd  only  near 
the  end  of  the  attack.  They  may  occasionally  be  met 
with  in  chronic  bronchitis  and  other  conditions.  Their 
nature  has  not  been  definitely  determined. 


Fig.  8. — Curschmann's  spirak:   I  Natural  size;  II  and  III,  enlarged:  a,  central  fiber 
(after  Curschmann). 


Macroscopically,  they  are  whitish  or  yellow,  twisted 
threads,  frequently  coiled  into  little  balls  (Fig.  8,  I). 
Their  length  is  rarely  over  1.5  cm.,  though  it  some- 
times exceeds  5  cm.  They  cannot  be  definitely  recog- 
nized with  the  naked  eye.  Under  a  i6-mm.  objective 
they  appear  as  mucous  threads  having  a  clear  central 
fiber,  about  which  are  wound  many  fine  fibrils  (Fig.  8, 


58 


THE   SPUTUM 


II  and  III).  Eosinophiles  are  usually  present  within 
them,  and  sometimes  Charcot-Leyden  crystals.  Not 
infrequently  the  spirals  are  imperfectly  formed,  con- 
sisting merely  of  twisted  strands  of  mucus  enclosing 
leukocytes.    The  central  fiber  is  absent  from  these. 

3.  Charcot=Leyden  Crystals. — Of  the  crystals  which 
may  be  found  in  the  sputum,  the  most  interesting  are  the 


Fig.  g. — Charcot-Leyden  crystals  (after  Riegel). 

Charcot-Leyden  crystals.  They  may  be  absent  when 
the  sputum  is  expectorated,  and  appear  in  large  numbers 
after  it  has  stood  for  some  time.  They  are  rarely  found 
except  in  cases  of  bronchial  asthma,  and  were  at  one 
time  thought  to  be  the  cause  of  the  disease.  The}- 
frequently  adhere  to  Curschmann  spirals.  Their  exact 
nature  is  unknown.  Their  formation  seems  to  be  in 
some  way  connected  with  the  presence  of  eosinophihc 


MICROSCOPIC   EXAMINATION  59 

cells.  Outside  of  the  sputum  they  are  found  in  the 
feces  in  association  with  animal  parasites,  and  in  the 
coagulated  blood  in  leukemia. 

They  are  colorless,  pointed,  often  needle-like  crystals 
(Fig.  9).  They  were  formerly  described  as  octahedral, 
but  are  now  known  to  be  hexagonal  in  cross-section. 
Their  size  varies  greatly,  the  average  length  being  about 
three  or  four  times  the  diameter  of  a  red  blood-corpuscle. 


Fig.  10. — Fibrinous  bronchial  cast  (Sahli). 

Other  crystals — hematoidin,  cholesterin,  and,  most 
frequently,  fatty-acid  needles  (see  Fig.  35) — are  common 
in  sputum  which  has  remained  in  the  body  for  a  consider- 
able time,  as  in  abscess  of  the  lung  and  bronchiectasis. 

4.  Bronchial  Casts. — These  are  casts  of  the  bronchi, 
frequently,  but  not  always,  composed  of  fibrin.  In 
color  they  are  usually  white  or  grayish,  but  may  be 
reddish  or  brown,  from  the  presence  of  blood-pigment. 


6o  THE  SPUTUM 

Their  size  varies  with  that  of  the  bronchi  in  which  they 
are  formed.  They  may,  rarely,  be  fifteen  or  more  centi- 
meters in  length.  When  large,  they  can  be  recognized 
with  the  naked  eye  by  floating  them  out  in  water  over 
a  black  surface;  when  small,  a  low  power  of  the  micro- 
scope must  be  used.  Their  branching,  tree-like  struc- 
ture (Fig.  lo)  is  usually  sufficient  to  identify  them. 

Fibrinous  casts  are  usually  indicative  of  fibrinous  or 
chronic  plastic  bronchitis,  but  may  also  be  found  in 


Fig.  II. — Sputum  from  a  case  of  actinomycosis;  stained  Qakob). 

diphtheria  of  the  smaller  bronchi.     Very  small  casts 
are  often  seen  in  croupous  pneumonia. 

5.  Actinomyces  Bovis  (Ray=fungus).— In  the  sputum 
of  pulmonary  actinomycosis  and  in  the  pus  from  actino- 
mycotic lesions  elsewhere  small,  yellowish,  "sulphur" 
granules  can  be  detected  with  the  unaided  eye.  With- 
out a  careful  macroscopic  examination  they  are  almost 
certain  to  be  overlooked.  The  fungus  can  be  seen  by 
crushing  one  of  these  granules  between  slide  and  cover, 


MICROSCOPIC  EXAMINATION  6l 

and  examining  with  a  low  power.  It  consists  of  a  net- 
work of  threads  having  a  more  or  less  radial  arrangement. 
In  cattle,  and  to  a  less  extent  in  man,  the  filaments  at 
the  periphery  of  the  nodule  present  club-shaped  extremi- 
ties (Fig.  ii).  It  can  be  brought  out  more  clearly  by 
running  a  little  solution  of  eosin  in  alcohol  and  glycerin 
under  the  cover.  This  organism,  also  called  Strepto- 
thrix  actinomyces,  apparently  stands  midway  between 
the  bacteria  and  the  molds.  It  stains  by  Gram's  method. 
Actinomycosis  of  the  lung  is  rare.  The  cUnical  pic- 
ture is  that  of  tuberculosis. 

6.  Molds  and  Yeasts. — The  hyphae  and  spores  of 
various  molds  are  occasionally  met  with  in  the  sputum. 
They  are  usually  the  result  of  contamination,  and  have 
little  significance.  The  hyphse  are  rods,  usually  jointed 
or  branched  (see  Fig.  62)  and  often  arranged  in  a  mesh- 
work  (mycelium) ;  the  spores  are  highly  refractive  spheres. 
Both  stain  well  with  the  ordinary  stains. 

In  the  extremely  rare  condition  of  systemic  blasto- 
mycosis the  specific  yeasts  have  been  found  in  the  sputimi 
in  large  numbers.  It  is  advisable  to  add  a  Httle  10  per 
cent,  caustic  soda  solution  and  examine  unstained. 

7.  Animal  Parasites. — These  are  extremely  rare  in 
the  sputum  in  this  country.  A  trichomonad,  perhaps 
identical  with  Trichomonas  vaginalis,  has  been  seen  in 
the  sputum  of  putrid  bronchitis  and  gangrene  of  the 
lung,  but  its  causal  relationship  is  doubtful.  In  Japan, 
infection  with  the  lung  fiukeworm,  Paragonimus  wes- 
termannii,  is  common,  and  the  ova  are  found  in  the  spu- 
tum. The  lung  is  not  an  uncommon  seat  for  echino- 
coccus  cysts,  and  booklets  and  scoHces  may  appear,  as 
may  also  Amoeba  histolytica,  when  a  hepatic  abscess  has 


$2  THE   SPUTUM 

ruptured  into  the  lung.  Larvae  of  Strongyloides  intes- 
Hnalis  and  of  the  hook-worm  have  been  reported.  Cil- 
iated body-cells,  with  cilia  in  active  motion,  are  not  in- 
frequently seen,  and  may  easily  be  mistaken  for  infusoria. 
All  the  above-mentioned  parasites  are  described  in 
Chapter  VI. 

B.   Stained  Sputum 

Structures  which  are  best  seen  in  stained  sputum  are 
bacteria  and  cells. 

A  number  of  smears  should  be  made  upon  sUdes  or 
covers.  These  films  must,  of  course,  be  thin,  but  it  is 
easily  possible  to  get  them  too  thin.  This  is  a  common 
error  of  students  who  have  just  finished  a  course  in 
bacteriology  and  who  have  there  been  accustomed  to 
work  with  scarcely  perceptible  films  of  bacteria.  It  is 
a  good  plan  to  sUde  off  the  cover-glass  from  the  prepara- 
tion used  for  the  unstained  microscopic  examinations. 
If  this  is  properly  done  satisfactory  smears  will  be  left 
on  both  slide  and  cover.  They  are  then  dried  in  the  air, 
and  fixed  in  the  flame,  as  described  on  page  466.  Fixation 
will  kiU  the  bacteria  when  covers  are  used,  and  the 
smears  may  be  kept  indefinitely;  but  smears  on  sUdes 
are  often  not  sterile,  and  should  be  handled  accord- 
ingly. One  of  the  smears  should  be  stained  with  some 
general  stain,  like  Loflier's  methylene  blue  or  pyronin- 
methyl  green  (see  p.  467) ,  which  will  give  a  good  idea  of 
the  various  cells  and  bacteria  present.  Special  stains  may 
then  be  applied,  as  indicated,  but  a  routine  examination 
should,  in  all  cases,  include  a  stain  by  the  method  for 
the  tubercle  bacillus  and  by  Gram's  method. 

1.  Bacteria. — Saprophytic  bacteria  from  mouth  con- 
tamination are  frequently  present  in  large  numbers  and 


MICROSCOPIC   EXAMINATION  63 

will  prove  confusing  to  the  inexperienced.  The  pres- 
ence of  squamous  cells  in  their  neighborhood  will  sug- 
gest their  source.  Among  the  pathogenic  organisms 
which  have  clinical  importance  are:  tubercle  bacilli; 
staphylococci  and  streptococci;  pneumococci;  bacilH  of 
Friedlander;  influenza  bacilH,  and  Micrococcus  catar- 
rhalis.  Their  cultural  characteristics  are  described  in 
Chapter  VIII. 

(i)  Tubercle  Bacillus. — The  presence  of  the  tubercle 
bacillus  may  be  taken  as  positive  evidence  of  the  ex- 
istence of  tuberculosis  somewhere  along  the  respiratory 
tract,  most  Ukely  in  the  lung.  In  laryngeal  tuberculosis 
it  is  not  easily  found  in  the  sputum,  but  can  fre- 
quently be  detected  in  swabs  made  directly  from  the 
larynx. 

Recognition  of  the  tubercle  bacillus  depends  upon  the 
fact  that  it  stains  with  difficulty;  but  that  when  once 
stained,  it  retains  the  stain  tenaciously,  even  when 
treated  with  a  mineral  acid,  which  quickly  removes  the 
stain  from  other  bacteria.  This  ''acid-fast"  property  is 
due  to  the  presence  of  a  waxy  or  lipoid  substance.  A 
number  of  the  best  staining  methods  are  included  here. 
Since  Gabbet's  method  is  probably  the  most  convenient 
for  general  purposes,  it  is  given  in  greater  detail  than 
the  others.  It  is  always  best  to  select  the  more  purulent 
portions  of  the  sputum,  keeping  away  from  the  mucoid 
parts.  If  bits  of  necrotic  tissue  are  present  they  may 
show  immense  numbers  of  tubercle  bacilli,  when  other 
portions  of  the  specimen  contain  very  few.  One  must, 
however,  be  on  his  guard  against  bits  of  food  which 
resemble  these  "caseous  particles."  The  specimen  should 
be  examined  while  fresh.    It  will  usually  liquefy  upon 


64  THE  SPUTUM 

standing,  and  this,  by  preventing  the  selection  of  parti- 
cles favorable  for  examination,  will  greatly  reduce  one's 
chances  of  finding  bacilli. 

Gabbet's  Method. — (i)  Spread  suspicious  particles  thinly 
and  evenly  upon  a  slide  or  a  cover-glass  held  in  the  grasp  of 
cover-glass  forceps.  In  general,  slides  are  more  satisfactory, 
but  cover-glasses  are  easier  to  handle  while  staining.  Do  not 
grasp  a  cover  too  near  the  edge  or  the  stain  will  not  stay 
on  it  well.  Tenacious  sputiun  will  spread  better  if  gently 
warmed  while  spreading. 

(2)  Dry  the  film  in  the  air. 

(3)  Fix  in  a  flame;  i.  e.,  pass  the  cover-glass  rather  slowly, 
with  film,  side  up,  three  times  (a  sUde  about  twelve  times) 
through  the  flame  of  a  Bursen  burner  or  alcohol  lamp  low 
down  in  the  flame.  Take  care  not  to  scorch.  A  scorched 
smear  has  a  brownish  tinge,  especially  at  the  edge.  The 
smear  can  likewise  be  fixed  by  flaming  with  alcohol  as  given 
for  blood-films  (see  p.  265).  Should  the  film  be  washed  off 
during  future  manipulations,  fixation  has  been  insufficient. 

(4)  Apply  as  much  carbol-fuchsin  as  will  stay  on,  and  hold 
over  a  flame  so  that  it  will  steam  for  three  minutes  or  longer, 
replacing  the  stain  with  a  dropper  as  it  evaporates.  If  the 
stain  is  allowed  to  evaporate  completely,  the  preparation  is 
ruined.  If  the  bacilli  are  well  stained  in  this  step,  there  will 
be  little  danger  of  decolorizing  them  later.  Too  great  heat 
will  interfere  with  the  staining  of  some  of  the  bacilli,  probably 
by  destroying  the  waxy  substance  upon  which  the  acid-fast 
property  depends.  It  is  better  to  stain  at  room  temperature 
for  twelve  to  twenty-four  hours,  although  this  is  not  often  done 
in  routine  work. 

(5)  Wash  the  film  in  water. 

(6)  Apply  Gabbet's  stain  to  the  imder  side  of  the  cover- 
glass  to  remove  excess  of  carbol-fuchsin,  and  then  to  the 


MICROSCOPIC   EXAMINATION  ,  65 

film  side.      Allow  this  to  act  for  one-fourth  to  one-half 
minute. 

(7)  Wash  in  water. 

(8)  If,  now,  the  thinner  portions  of  the  film  are  blue,  pro- 
ceed to  the  next  step;  if  they  are  still  red,  repeat  steps  (6) 
and  (7)  until  the  red  has  disappeared.  Too  long  application 
of  Gabbet's  stain  will  decolorize  the  tubercle  bacilli. 

(9)  Place  the  preparation  between  layers  of  filter-paper 
and  dry  by  rubbiiig  with  the  fingers,  as  one  would  in  blotting 
ink. 

(10)  Put  a  drop  of  Canada  balsam  upon  a  clean  slide,  place 
the  cover-glass  film  side  down  upon  it,  and  examine  with  an 
immersion  objective.  Cedar  oil  or  water  may  be  used  in 
place  of  balsam  for  temporary  preparations.  Smears  on 
slides  may  be  examined  directly  with  an  oil-immersion  lens, 
no  cover  being  necessary. 

Carbol-fuchsin  is  prepared  by  mixing  10  c.c.  of  a  saturated 
alcoholic  solution  of  fuchsin  with  90  c.c.  of  5  per  cent,  aqueeus 
solution  of  phenol.  This  stain  sometimes  rather  suddenly 
"goes  bad,"  in  which  case  it  appears  more  transparent  than 
normal  and  the  bottle  contains  a  dark  red  sediment. 

Gabbet's  stain  consists  of  methylene  blue,  2  gm.;  25  per 
cent,  sulphuric  acid,  100  c.c. 

Both  stains  can  be  purchased  ready  prepared. 

Ziehl-Neelson  Method. — The  objection  is  often  made  to 
the  above  method  that  decolorization  is  masked  by  the  blue 
in  Gabbet's  stain.  Although  this  will. not  make  trouble  if 
step  (8)  is  carefully  carried  out,  most  experienced  workers 
prefer  the  Ziehl-Neelson  method.  This  resembles  Gabbet's 
method,  with  the  following  exceptions:  After  the  staining 
with  carbol-fuchsin  the  smear  is  washed  in  5  per  cent,  nitric 
acid  (or,  better,  a  mixture  of  3  c.c.  concentrated  hydrochloric, 
acid  and  97  c.c.  70  per  cent,  alcohol)  until  decolqirized, 
washed  in  water,  stained  lightly  with  Loflfler's  methylene 
blue,  again  washed,  and  finally  dried  and  mounted. 
5 


66  THE   SPUTUM 

Pappenheim's  Method. — This  is  the  same  as  Gabbet's 
method,  except  that  Pappenheim's  methylene-blue  solution 
is  substituted  for  Gabbet's  stain.    This  consists  of: 

Corallin  (rosolic  acid) ' i  gm; 

Absolute  alcohol loo  c.c. 

Saturate  with  methylene  blue  and  add  20  c.c.  glycerin. 

The  method  is  very  satisfactory  for  routine  work.  De- 
colorization  of  the  tubercle  bacillus  is  practically  impos- 
sible: it  retains  its  red  color,  even  when  soaked  overnight 
in  Pappenheim's  solution.  The  stain  was  originally  recom- 
mended as  a  means  of  differentiating  the  smegma  bacillus, 
which  is  decolorized  by  it;  but  it  is  not  to  be  absolutely  relied 
upon  for  this  purpose. 

In  fihns  stained  by  these  methods  tubercle  bacilli, 
if  present,  will  be  seen  as  slender  red  rods  upon  a  blue 
background  of  mucus  and  cells  (Plate  II,  Fig.  2).  They 
vary  considerably  in  size,  averaging  3  to  4  /w  in  length — 
about  one-half  the  diameter  of  a  red  blood-corpuscle. 
Beginners  must  be  warned  against  mistaking  the  edges 
of  cells,  or  particles  which  have  retained  the  red  stain, 
for  bacilli.  The  appearance  of  the  bacilli  is  almost  always 
typical,  and  if  there  seems  room  for  doubt,  the  structure 
in  question  is  probably  not  a  tubercle  bacillus.  They 
may  lie  singly  or  in  groups.  They  are  very  frequently 
bent  and  often  have  a  beaded  appearance.  It  is  possible 
that  the  larger,  beaded  bacilli  indicate  a  less  active 
tuberculous  process  than  do  the  smaller,  uniformly 
stained  ones.  Sometimes  they  are  present  in  great  num- 
bers— thousands  in  a  field  of  the  2-mm.  objective.  Some- 
times, even  in  advanced  cases,  several  cover-glasses  must 
be  examined  to  find  a  single  bacillus.    At  times  they  are 


PLATE  II 


Fig.   I. — Heart-failure  cells  in  sputum,  containing  blood-pigment,  from 
a  case  of  cardiac  congestion  of  the  lungs  (Jakob). 


Fig.  2. — A,  Sputum  showing  tubercle  bacilli  stained  with  car- 
bolfuchsin  and  Gabbet's  methylene-blue  solution  (obj.  one-twelfth 
oil-immersion);  B,  sputum  of  anthracosis,  showing  particles  of  coal-dust 
stained  with  methylene-blue  (obj.  one-twelfth  oil-immersion)  (Boston). 


MICROSCOPIC   EXAMINATION  67 

SO  few  that  none  are  found  in  stained  smears,  and  special 
methods  are  required  to  detect  them.  The  number  may 
bear  some  relation  to  the  severity  of  the  disease,  but  this 
relation  is  by  no  means  constant.  The  mucoid  sputum 
from  an  incipient  case  sometimes  contains  great  num- 
bers, while  sputum  from  large  tuberculous  cavities  at 
times  contains  very  few.  Failure  to  find  them  is  not 
conclusive,  though  their  absence  is  much  more  signifi- 
cant when  the  sputum  is  purulent  than  when  it  is  mucoid. 
When  it  is  desired  to  record  the  approximate  number 
of  bacilli  present,  the  Gaffky  table  as  modified  by  Brown 
may  be  employed,  using  an  oil-immersion  lens  and  4X 
ocular : 

I.  One  to  four  bacilli  to  the  slide. 
II.  Average  of  one  in  many  fields. 
III.  Average  of  one  in  a  field. 
rV.  Average  of  two  to  three  in  a  field. 
V.  Average  of  four  to  six  in  a  field. 
VI.  Average  of  seven  to  twelve  in  a  field. 
VII.  Average  of  thirteen  to  twenty-five  in  a  field. 
VIII.  Average  of  about  fifty  in  a  field. 
IX.  Average  of  about  one  hundred  in  a  field. 
X.  Enormous  numbers  in  a  field. 

Since  the  sputum  raised  at  various  times  in  the  day, 
and  even  different  parts  of  the  same  sample,  may  vary 
greatly  in  bacillary  content,  such  a  table  is  of  little  value 
unless  the  twenty-four-hour  sputum  is  collected  and  uni- 
formly mixed  before  preparing  the  slides.  This  is  satis- 
factorily accomplished  by  the  antiformin  method. 

When  bacilli  are  not  found  in  suspicious  cases,  one  of 
the  following  methods  should  be  tried: 


68  THE  SPUTUM 

(i)  Antifonnin  Method. — ^This  has  lately  come  into  use, 
and  has  superseded  the  older  methods  of  concentration. 
The  chief  diflSculty  with  the  older  methods,  such  as  boiling 
with  caustic  soda,  is  that  the  bacilli  are  so  injured  in  the 
process  that  they  do  not  stain  characteristically. 

Antiformin  is  a  trade  name  for  a  preparation  consisting 
essentially  of  equal  parts  of  a  15  per  cent,  solution  of  caustic 
soda  and  a  20  per  cent,  solution  of  sodium  hypochlorite.  It 
keeps  fairly  well.  Substitutes  appear  to  be  less  satisfactory 
than  the  original  preparation. 

LbflBier's  method  is  probably  the  best  for  clincial  work. 
It  kills  the  bacilli,  so  that  there  is  no  danger  in  handling 
the  material.  Upon  this  account,  however,  it  is  not  appli- 
cable to  isolation  of  tubercle  bacilli  for  pure  cultures. 

Place  10  to  20  c.c.  of  the  sputum  in  a  small  flask,  with 
an  equal  amount  of  50  per  cent,  antiformin,  and  heat  to 
the  boiling-point.  The  sputum  will  be  thoroughly  lique- 
fied, usually  within  a  few  seconds.  For  each  10  c.c.  of 
the  resulting  fluid  add  1.5  c.c.  of  a  mixture  of  i  volimie  of 
chloroform  and  9  volumes  of  alcohol.  Shake  vigorously  for 
several  minutes  or  until  emulsification  has  taken  place.  The 
object  is  to  impregnate  the  lipoid  capsule  of  the  bacilli  with 
chloroform,  thus  increasing  their  specific  gravity.  Pour  off 
the  emulsion  into  centrifuge  tubes  and  centrifugalize  at  high 
spefed  for  about  fifteen  minutes.  The  chloroform  will  go  to 
the  bottom,  and  the  sediment  which  collects  on  its  surface 
in  a  thin  firm  layer  will  contain  the  tubercle  bacilli.  Pour 
off  the  supernatant  Uquid  and  transfer  the  sediment  to  glass 
slides,  removing  the  excess  of  fluid  with  filter-paper.  To 
facilitate  removal  of  the  disk  of  sediment  in  toto  Williamson 
recommends  the  use  of  a  centrifuge  tube,  the  lower  §  inch 
of  which  is  of  uniform  caliber  and  the  bottom  of  which  is 
open  and  plugged  with  a  rubber  stopper.  Add  a  little  egg- 
albumen  solution  (see  p.  72)  or,  better,  some  of  the  original 
sputum,  to  cause  the  film  to  adhere  to  the  slide,  mix  well. 


MICROSCOPIC  EXAMINATION  69 

spread  into  a  uniform  layer,  and  finally  dry,  fix,  and  stain  by 
the  Ziehl-Neelson  method.  Loffler  recommends  o.i  per  cent, 
solution  of  malachite  green  for  counterstain. 

(2)  Animal  Inoculation. — Inoculation  of  guinea-pigs  is 
the  court  of  last  appeal  in  detection  of  tubercle  bacilli.  The 
method  is  described  on  p.  375. 

There  are  a  number  of  bacilli,  called  acid-fast  bacilli, 
which  stain  in  the  same  way  as  the  tubercle  bacillus. 
They  stain  with  difficulty,  and  when  once  stained,  retain 
the  color  even  when  treated  with  a  mineral  acid;  but, 
unlike  the  tubercle  bacillus,  most  of  them  can  be  decolor- 
ized with  alcohol.  Of  these,  the  smegma  bacillus  is  the 
only  one  likely  ever  to  cause  confusion.  It,  or  a  similar 
bacillus,  is  sometimes  found  in  the  sputum  of  gangrene 
of  the  lung.  It  occurs  normally  about  the  glans  penis 
and  the  clitoris,  and  is  often  present  in  the  urine  and 
in  the  wax  of  the  ear.  The  method  of  distinguish- 
ing it  from  the  tubercle  bacillus  is  given  later  (see 
p.  205). 

Other  bacteria  than  the  acid-fast  group  are  stained 
blue  by  Gabbet's  and  the  Ziehl-Neelson  method.  Those 
most  commonly  found  are  staphylococci,  streptococci, 
and  pneumococci.  Their  presence  in  company  with  the 
tubercle  bacillus  constitutes  mixed  infection,  which  is 
much  more  serious  than  single  infection  by  the  tubercle 
bacillus.  It  is  to  be  remembered,  however,  that  a  few 
of  these  bacteria  may  reach  the  sputum  from  the  upper 
air-passages.  CHnically,  mixed  infection  is  evidenced  by 
fever. 

.  Within  the  past  few  years  much  interest  has  centered 
in  the  so-called  "Much  granules."  These  are  Gram- 
positive  but  non-acid-fast  granules  and  rods;  and  are 


70  THE   SPUTUM 

apparently  forms  of  the  tubercle  bacillus,  since  material 
containing  them  causes  tuberculosis  when  injected  into 
guinea-pigs.  They  may  be  present  either  alone  or  in 
company  with  the  ordinary  acid-fast  form.  Their 
recognition  accounts  for  the  well-known  fact  that  in 
some  cases  of  undoubted  pulmonary  tuberculosis  no 
tubercle  bacilli  have  been  demonstrated  in  the  sputxun. 

Methods  for  Much  Granules. — (i)  Stain  the  dried  and 
fixed  film  with  carbol-gentian  volet,  heating  to  steaming 
three  times. 

(2)  Rinse  with  water  and  apply  Gram's  iodin  solution  for 
five  minutes. 

(3)  Wash  in  water  and  apply  successively  5  per  cent, 
nitric  acid  one  minute,  3  per  cent,  hydrochloric  acid  ten 
seconds,  equal  parts  of  absolute  alcohol  and  acetone  until 
color  ceases  to  come  off. 

(4)  Wash  in  water  and  counterstain  with  an  aqueous  solu- 
tion of  saffranin. 

(5)  Wash,  dry,  and  mount. 

Tubercle  bacilli  and  Much  granules  will  be  purple;  other 
organisms,  red. 

Perhaps  more  satisfactory  is  a  combination  of  the  Ziehl- 
Neelson  and  Gram's  method  as  advocated  by  Pontes:  After 
staining  with  carbol-fuchsin  and  decolorizing  (see  p.  65),  the 
preparation  is  stained  by  Gram's  method  (see  p.  467)  and 
counterstained  with  Bismarck  brown., 

Carbol-gentian  violet  consists  of  saturated  alcoholic  solu- 
tion of  gentian-violet  10  c.c;  2  per  cent,  phenol,  90  c.c. 

(2)  Staphylococcus  and  Streptococcus  (see  p.  368). — 
One  or  both  of  these  organisms  is  commonly  present  in 
company  with  the  tubercle  bacillus  in  the  sputum  of 
advanced  phthisis  (Plate  II,  Fig.  2).     They  are  often 


MICROSCOPIC   EXAMINATION  7 1 

found  in  bronchitis,  catarrhal  pneumonia,   and  many 
other  conditions. 

(3)  Pneumococcus  (Diplococcus  of  Frankel). — The 
pneumococcus  is  the  causative  agent  in  nearly  all  cases 
of  croupous  pneumonia,  and  is  commonly  found  in  large 
numbers  in  the  rusty  sputum  of  this  disease.  It  is  some- 
times met  with  in  the  sputum  of  catarrhal  pneumonia, 
bronchitis,  and  tuberculosis.    It  is  also  an  important  fac- 


Fig.  12. — Diplococcus  pnevimonix  in  the  blood  (Xiooo)  (Frankel  and  Pfeiffer). 

tor  in  the  causation  of  pleurisy,  meningitis,  otitis  media, 
and  other  inflammations.  It  is  frequently  present  in 
the  saliva  in  health.  Pneumococci  are  about  the  size 
of  streptococci.  They  are  ovoid  in  shape,  and  lie  in 
pairs,  end  to  end,  often  forming  short  chains.  Each  is' 
surrounded  by  a  gelatinous  capsule,  which  is  its  dis- 
tinctive feature  (Fig.  12). 

The  pneumococcus  is  closely  related  to  the  strepto- 
coccus, and  it  is  sometimes  extremely  difficult  to  differ- 


72  THE  SPUTUM 

entiate  them  even  by  culture  methods  (for  which  see 
p.  420).  The  morphology  of  the  pneumococcus,  the 
fact  that  it  is  Gram-positive,  and  the  presence  of  a  cap- 
sule are,  however,  generally  sufficient  for  its  recognition 
in  smears  from  sputum  or  pus.  The  capsule  is  often 
seen  as  a  halo  around  pairs  of  cocci  in  smears  stained 
by  the  ordinary  methods,  particularly  Gram's  method, 
but  to  show  it  well  special  methods  are  required.  There 
are  numerous  special  methods  of  staining  capsules  which 
are  applicable  to  other  encapsulated  bacteria,  as  well  as 
to  the  pneumococcus,  but  few  of  them  are  satisfactory. 
Buerger's  method  can  be  recommended.  It  is  especially 
useful  with  cultures  upon  serum  media,  but  is  applicable 
also  to  the  sputum.  Smith's  method  is  easier  of  ap- 
plication, and  apparently  gives  uniformly  good  results. 
The  India-ink  method  described  for  the  organism  of 
syphilis  is  likewise  said  to  show  capsules  satisfactorily. 
The  sputum  should  be  fresh — not  more  than  three  or 
four  hours  old. 

Buerger's  Method  for  Capsules. — (i)  Mix  a  few  drops 
each  of  the  sputum  and  blood-serum  or  egg-albumen  solu- 
tion (egg-albiunen,  distilled  water,  equal  parts;  shake,  filter 
through  cotton,  and  add  about  0.5  per  cent,  phenol).  Blood- 
serum  can  be  obtained  as  described  for  the  Widal  test  (see 
p.  498).  Make  thin  smears  from  the  mixture,  and  just  as  the 
edges  begin  to  dry,  cover  with  Miiller's  fluid  (potassium  di- 
chromate,  2.5  gm.;  sodium  sulphate,  i.o  gm. ;  water,  100  c.c.) 
saturated  with  mercuric  chlorid  (ordinarily  about  5  per  cent.). 
Gently  warm  over  a  flame  for  about  three  seconds. 

(2)  Rinse  very  quickly  in  water. 

(3)  Flush  once  with  alcohol. 

(4)  Apply  tincture  of  iodin  for  one  to  two  minutes. 


MICROSCOPIC  EXAMINATION  73 

(5)  Thoroughly  wash  off  the  iodin  with  alcohol  and  dry  in 
the  air. 

(6)  Stain  about  three  seconds  with  weak  anilin-gentian 
violet  freshly  made  up  as  follows:  Anilin  oil,  10;  water,  100; 
shake;  filter;  and  add  5  c.c.  of  a  saturated  alcoholic  solution 
of  gentian  violet. 

(7)  Rinse  off  the  stain  with  2  per  cent,  solution  of  sodium 
chlorid,  mount  in  this  solution,  and  examine  with  a  one- 
twelfth  objective. 

Buerger  suggests  a  very  useful  variation  as  follows:  After 
the  alcohol  wash  and  drying,  the  specimen  is  stained  by 
Gram's  method  (see  p.  467),  counterstained  with  aqueous 
solution  of  fuchsin,  washed,  and  mounted  in  water.  The 
pneumococcus  holds  the  purple  stain,  while  all  capsules  take 
the  pink  counters  tain. 

Smith's  Method. — (i)  Make  thin  smears  of  the  sputum 
or  other  material,  which  should  be  as  fresh  as  possible. 

(2)  Fix  in  the  flame  in  the  usual  manner. 

(3)  Apply  a  10  per  cent,  aqueous  solution  of  phospho- 
molybdic  acid  (Merck)  for  four  to  five  seconds. 

(4)  Rinse  in  water. 

(5)  Apply    anilin-gentian    violet,    steaming    gently    for 
fifteen  to  thirty  seconds. 

(6)  Rinse  in  water. 

(7)  Apply  Gram's  iodm  solution,  steaming  gently  for 
fifteen  to  thirty  seconds. 

(8)  Wash  in  95  per  cent,  alcohol  until  the  purple  color 
ceases  to  come  off. 

(9)  Rinse  in  water. 

(10)  Apply  a  6  per  cent,  aqueous  solution  of  eosin  (Grli- 
bler,  w.  g.),  and  gently  warm  for  one-half  to  one  minute. 

(11)  Rinse  in  water. 

-    (12)  Wash  in  absolute  alcohol. 

(13)  •  Clear  in  xylol. 

(14)  Mount  in  balsam. 


74  THE   SPUTUM 

This  is  essentially  Gram's  method  (see  p.  467),  preceded 
by  treatment  with  phosphomolybdic  acid  and  followed  by 
eosin.  Gram-positive  bacteria  like  the  pneumococcus  are 
deep  purple;  capsules  are  pink  and  stand  out  clearly. 

When  the  method  is  applied  to  Gram-negative  bacteria, 
steps  5  to  9  inclusive  are  omitted;  and  between  steps  11  and 
12  the  preparation  is  counterstained  with  Loffler's  methylene 
blue,  gently  warming  for  fifteen  to  thirty  seconds. 

Anilin-gentian  violet. — Ehrlich's  formula  is  the  one  gener- 
ally used,  but  this  keeps  only  a  few  weeks.  Stirling's  solu- 
tion, which  keeps  much  better  and  seems  to  give  equal  results, 
is  as  follows:  gentian  violet,  5  gm.;  alcohol,  10  c.c. ;  anilin 
oil,  2  c.c. ;  water,  88  c.c. 

Formalin-gentian  violet  is  a  satisfactory  substitute  for 
anilin-gentian  violet  and  is  permanent.  It  consists  of  5  per 
cent,  solution  formalin,  75  parts;  saturated  alcoholic  solu- 
tion gentian  violet,  25  parts. 

Gramas  lodin  Solution. — lodin,  i  gm.;  potassium  iodid, 
2  gm.;  water,  300  c.c. 

Loffler's  alkaline  methylene-hlue  is  a  very  generally  useful 
stain  for  bacteria.  It  is  composed  of  30  parts  of  a  saturated 
alcoholic  solution  of  methylene  blue  and  100  parts  of  a 
I  :  10,000  aqueous  solution  of  caustic  potash.  It  keeps 
indefinitely. 

(4)  Bacillus  of  Friedlander  (Bacillus  mucosus  cap- 
sulatus). — In  a  small  percentage  of  cases  of  pneumonia 
this  organism  is  found  alone  or  in  company  with  the 
pneumococcus.  Its  pathologic  significance  is  uncer- 
tain. It  is  often  present  in  the  respiratory  tract  under 
normal  conditions.  Friedlander's  bacilli  are  non-motile, 
encapsulated  rods,  sometimes  arranged  in  short  chains 
(Fig.   13).     Very  short  individuals  in  pairs  closely  re- 


MICROSCOPIC   EXAMINATION 


7S 


semble  pneumococci,  from  which  they  are  distinguished 
by  the  fact  that  they  are  Gram-decolorizing. 

(5)  Bacillus  of  Influenza. — This 
is  the  etiologic  factor  in  true  in- 
fluenza, although  conditions  which 
are  cUnically  similar  or  identical 
may  be  caused  by  the  pneumococ- 
cus,  streptococcus,  or  Micrococcus 
catarrhalis.  It  is  present,  often  in 
large  numbers,  in  the  nasal  and 
bronchial  secretions,  and  is  also 
found  in  the  local  lesions  following 
influenza.  Chronic  infection  by  influenza  bacilli  may 
be  mistaken  clinically  for  tuberculosis,  and  they  should 
be  searched  for  in  all  cases  of  obstinate  chronic  bron- 
chitis. 


Fig.  I..,,  i  :.....„..  „;  i  ba- 
cillus in  pus  from  pulmonary 
abscess  (one-twelfth  objective) 
(Boston). 


Fig.  14. — Bacillus  of  influenza;  cover-glass  preparation  of  sputum  from  a  case  of  influenza, 
showing  the  bacilli  in  leukocytes;  highly  magnified  (Pfeiffer). 


Their  recognition  depends  upon  the  facts  that  they 
are  extremely  small  bacilli;  that  most  of  them  lie  within 


76  THE  SPUTUM 

the  pus-cells;  that  their  ends  stain  more  deeply  than  their 
centers,  sometimes  giving  the  appearance  of  minute 
diplococci;  and  that  they  are  decolorized  by  Gram's 
method  of  staining  (Figs.  14  and  157). 

They  are  well  stained  by  dilute  fuchsin  or  by  Pappen- 
heim's  pyronin-methyl  green,  but  are  more  certainly 
recognized  by  Gram's  method  with  the  pyronin-methyl 
green  for  counterstain. 

(6)  Bacillus  pertussis. — The  whooping-cough  bacillus 
is  a  minute,  ovoid.  Gram-negative  bacillus  which  stains 
feebly  with  the  ordinary  dyes,  and  sometimes,  though 
not  usually,  Ues  within  pus  cells.  It  can  be  demonstrated 
by  the  method  given  for  the  influenza  bacillus. 

(7)  Micrococcus  catarrhalis. — This  organism  is  fre- 
quently present  in  the  sputum  in  inflammatory  condi- 
tions of  the  respiratory  tract  resembling  influenza. 
It  is  sometimes  present  in  the  nasal  secretions  in  health. 
It  is  a  Gram-negative  diplococcus,  frequently  intra- 
cellular, and  can  be  distinguished  from  the  meningo- 
coccus and  gonococcus  only  by  means  of  cultures.  The 
staining  method  recommended  for  the  influenza  bacillus 
is  best.    It  grows  readily  on  ordinary  media. 

2.  Cells. — These  include  pus-corpuscles,  epithelial 
cells,  and  red  blood-corpuscles. 

(i)  Pus-corpuscles  are  present  in  every  sputum,  and 
at  times  the  sputum  may  consist  of  little  else.  They  are 
the  polymorphonuclear  leukocytes  of  the  blood,  and 
appear  as  rounded  cells  with  several  nuclei  or  one  very 
irregular  nucleus  (Fig.  11  and  Plate  II,  Fig.  2).  They 
are  frequently  filled  with  granules  of  coal-dust  and  are 
often  much  degenerated.  Such  coal-dust-laden  leuko- 
cytes  are   especially   abundant   in   anthracosis,   where 


MICROSCOPIC   EXAMINATION  77 

angular  black  particles,  both  intra-  and  extra-cellular, 
are  often  so  numerous  as  to  color  the  sputum  (Plate  II, 
Fig.  2,  B).  Occasionally  mononuclear  leukocytes  are 
present. 

Eosinophilic  cells  are  quite  constantly  found  in  large 
numbers  in  the  sputum  of  bronchial  asthma  near  the 
time  of  the  paroxysm,  and  constitute  one  of  the  most 
distinctive  features  of  the  sputum  of  this  disease.    They 


Fig.  IS. — Sputum  from  a  case  of  asthma  showing  leukocytes,  some  containing  eosino- 
philic granules;  free  eosinophilic  granules  and  micrococci;  stained  with  eosin  and  methy- 
lene blue  (X  350)  (Jakob). 

resemble  ordinary  pus-corpuscles,  except  that  their 
cytoplasm  is  filled  with  coarse  granules  having  a  marked 
affinity  for  eosin.  It  is  worthy  of  note  that  many  of 
them,  sometimes  the  majority,  are  mononuclear.  Large 
numbers  of  free  granules,  derived  from  disintegrated 
cells,  are  also  found  (Fig.  15). 

Ordinary  pus-cells  are  easily  recognized  in  sputum 
stained  by  any  of  the  methods  already  given.  For 
eosinophilic  cells,  some  method  which  includes  eosin  must 


78 


THE   SPUTUM 


be  used.  A  simple  method  is  to  stain  the  dried  and  fixed 
fihn  two  or  three  minutes  with  saturated  solution  of 
eosin,  and  then  one-haK  to  one  minute  with  Loffler's 
methylene  blue;  nuclei  and  bacteria  will  be  blue,  eosino- 
philic granules  bright  red. 

(2)  Epithelial  cells  may  come  from  any  part  of  the 
respiratory  tract.    A  few  are  always  present,  since  des- 


Fig.  16. — Different  morphologic  elements  of  the  sputum  (unstained):  a,  b,  c,  Pulmo- 
nary or  alveolar  epithelium — a,  with  normal  lung  pigment  (carbon);  b,  with  fat-droplets; 
c,  with  myelin  globules;  d,  pus-corpuscles;  e,  red  blood -corpuscles;  /.  cylindric  beaker- 
shaped  bronchial  epithelial  cells;  g,  free  myelin  globules;  h,  ciliated  epithelium  of  diflFerent 
kinds  from  the  nose,  altered  by  coryza;  t,  squamous  cells  from  the  pharynx  (after  Bizzo- 
zero). 


quamation  of  cells  goes  on  constantly.  Their  recogni- 
tion is  important  chiefly  as  an  aid  in  deciding  upon  the 
source  of  the  portion  of  the  sputum  in  which  they  are 
found.  In  suspected  lung  conditions  it  is  manifestly 
useless  to  study  material  from  the  nose  only,  yet  this 


MICROSCOPIC   EXAMINATION  79 

is  not  infrequently  done.  They  have  little  diagnostic 
value,  although  a  considerable  excess  would  indicate  a 
pathologic  condition  at  the  site  of  their  origin.  Any 
of  the  stains  mentioned  above  will  show  them,  and  they 
can  usually  be  identified  in  unstained  sputum.  In 
general,  three  forms  are  found: 

(j)  Squamous  Cells. — Large,  flat,  polygonal  cells  with 
a  comparatively  small  nucleus  (Fig.  i6,  i).  They  come 
from  the  upper  air-passages,  and  ^re  especially  numerous 
in  laryngitis  and  pharyngitis.  They  are  frequently 
studded  with  bacteria — most  commonly  diplococci. 

{h)  Cylindric  Cells  from  the  Nose,  Trachea,  and  Bronchi 
(Fig.  16,  /,  h). — These  are  not  usually  abundant,  and, 
as  a  rule,  they  are  not  identified  because  much  altered 
from  their  original  form,  being  usually  round  and  swollen. 
When  very  fresh,  they  may  retain  their  cylindric  form, 
sometimes  bearing  cilia  in  active  motion. 

(c)  Alveolar  Cells. — Rather  large,  round,  or  oval  cells 
with  one  or  two  round  nuclei  (Fig.  16).  Their  source  is 
presumably  the  pulmonary  alveoli.  Like  the  leuko- 
cytes, they  frequently  contain  particles  of  carbon  (nor- 
mal lung  pigment).  In  chronic  heart  disease,  owing  to 
long-continued  passive  congestion,  they  may  be  filled 
with  brown  granules  of  altered  blood-pigment,  and  are 
then  called  "heart-failure  cells."  The  presence  of  these 
cells  in  considerable  numbers,  by  directing  one's  atten- 
tion to  the  heart,  will  sometimes  clear  up  the  etiology 
of  a  chronic  bronchitis.  They  are  best  seen  in  unstained 
sputum,  appearing  as  grayish  or  colorless  balls  filled  with 
rounded  granules  of  brown  or  yellow  pigment.  (See 
Plate  II,  Fig.  i.)  Such  cells  are  also  present  after  pul- 
monary hemorrhage. 


8o  THE   SPUTUM 

Alveolar  cells  commonly  contain  fat-droplets  and,  less 
frequently,  myelin  globules.  The  latter  are  colorless, 
rounded  bodies,  sometimes  resembling  fat-droplets,  but 
often  showing  concentric  or  irregularly  spiral  markings 
(Fig.  i6,  c,  g).  They  are  also  found  free  in  the  sputum. 
They  are  abundant  in  the  scanty  morning  sputum  of 
apparently  healthy  persons,  but  may  be  present  in  any 
mucoid  sputum. 

(3)  Red  blood-corpuscles  may  be  present  in  small 

numbers  in  almost  any  sputum.    When  fairly  constantly 

present  in  considerable  numbers,  they  are  suggestive  of 

phthisis.    The  corpuscles,  when  fresh,  are  shown  by  any 

of  the  staining  methods  which  include  eosin.    They  are 

commonly  so  much  degenerated  as  to  be  unrecognizable, 

and  often  only  altered  blood-pigment  is  left.    Ordinarily, 

blood  in  the  sputum  is  sufficiently  recognized  with  the 

naked  eye. 

nL  CHEMIC  EXAMINATION 

There  is  little  to  be  learned  from  a  chemic  examina- 
tion, and  it  is  rarely  undertaken.  Recently,  however, 
it  has  been  shown  that  the  presence  or  absence  of  albumin 
may  have  clinical  significance.  Albumin  is  constantly 
present  in  the  sputum  in  pneumonia,  pulmonary  edema, 
and  tuberculosis.  It  is  usually  absent  in  bronchitis. 
A  test  for  albumin  may,  therefore,  be  of  great  value  in 
distinguishing  between  bronchitis  and  tuberculosis,  a 
negative  result  practically  proving  the  absence  of  tuber- 
culosis. It  is  carried  out  as  follows:  The  sputum  is 
acidified  with  acetic  acid  to  precipitate  mucin  and  fil- 
tered. If  tenacious,  it  is  first  shaken  up  with  water. 
The  filtrate  is  then  tested  for  albumin,  as  described  in 
the  chapter  upon  the  Urine.    Active  cases  of  phthisis, 


THE   SPUTUM  IN  DISEASE  8 1 

whether  early  or  far  advanced,  generally  show  0.2  per 
cent,  or  more  albumin;  slightly  active  cases,  less  than 
0.2  per  cent.  The  sputmn  must  be  fresh,  otherwise  a 
negative  reaction  may  have  changed  to  positive. 

IV.  THE  SPUTUM  IN  DISEASE 

Strictly  speaking,  any  appreciable  amount  of  sputum 
is  abnormal.  A  great  many  healthy  persons,  however, 
raise  a  small  quantity  each  morning,  owing  chiefly  to 
the  irritation  of  inhaled  dust  and  smoke.  Although 
not  normal,  this  can  hardly  be  spoken  of  as  pathologic. 
It  is  particularly  frequent  in  city  dwellers  and  in  those 
who  smoke  cigarettes  to  excess.  In  the  latter  the 
amount  is  sometimes  so  great  as  to  arouse  suspicion  of 
tuberculosis.  Such  "normal  morning  sputum"  gen- 
erally consists  of  small,  rather  dense,  mucoid  masses, 
translucent  white,  or,  when  due  to  inhaled  smoke,  gray 
in  color.  Microscopically,  there  are  a  few  pus-cor- 
puscles, and,  usually,  many  alveolar  cells,  both  of  which 
may  contain  carbon  particles.  The  alveolar  cells  com- 
monly show  myehn  degeneration,  and  free  myelin  glob- 
ules may  be  present  in  large  numbers.  Saprophytic 
bacteria  may  be  present,  but  are  not  abundant. 

1 .  Acute  Bronchitis. — There  is  at  first  a  small  amount 
of  tenacious,  almost  purely  mucoid  sputum,  frequently 
blood  streaked.  This  gradually  becomes  more  abun- 
dant, mucopurulent  in  character,  and  yellowish  or  gray 
in  color.  At  first  the  microscope  shows  a  few  leukocytes 
and  alveolar  and  bronchial  cells;  later  the  leukocytes 
become  more  numerous.  Bacteria  are  not  usually 
abundant. 

2.  Chronic  Bronchitis. — The  sputum  is  usually  abun- 

6 


82  THE   SPUTUM 

dant,  mucopurulent,  and  yellowish  or  yellowish-green  in 
color.  Nummular  masses  like  those  of  tuberculosis  are 
sometimes  seen.  Microscopically,  there  are  great  num- 
bers of  leukocytes,  often  much  degenerated.  Epithe- 
lium is  not  abundant.  Bacteria  of  various  kinds,  espe- 
cially staphylococci,  are  usually  numerous. 

In  fibrinous  bronchitis  there  are  found,  in  addition, 
fibrinous  casts,  usually  of  medium  size. 

In  the  chronic  bronchitis  accompanying  long-continued 
passive  congestion  of  the  lungs,  as  in  poorly  compensated 
heart  disease,  the  sputum  may  assume  a  rusty  brown 
color,  owing  to  presence  of  large  numbers  of  the  "heart- 
failure  cells"  previously  mentioned. 

3.  Bronchiectasis. — When  there  is  a  single  large 
cavity,  the  sputum  is  very  abundant  at  intervals, — 
sometimes  as  high  as  a  liter  in  twenty-four  hours, — and 
has  a  very  offensive  odor.  It  is  thinner  than  that  of 
chronic  bronchitis,  and  upon  standing  separates  into 
three  layers  of  pus,  mucus,  and  frothy  servun.  It  contains 
great  numbers  of  miscellaneous  bacteria. 

4.  Gangrene  of  the  Lung.— The  sputum  is  abun- 
dant, fluid,  very  offensive,  and  brownish  in  color.  It 
separates  into  three  layers  upon  standing — a  brown 
deposit,  a  clear  fluid,  and  a  frothy  layer.  Microscopic- 
ally, few  cells  of  any  kind  are  found.  Bacteria  are  ex- 
tremely numerous;  among  them  may  sometimes  be 
found  an  acid-fast  bacillus  probably  identical  with  the 
smegma  bacillus.  As  before  stated,  elastic  fibers  are 
usually  present  in  large  fragments. 

5.  Pulmonary  Edema. — Here  there  is  an  abundant, 
watery,  frothy  sputum,  varying  from  faintly  yellow  or 
pink   to  dark  brown  in  color;   a  few  leukocytes  and 


THE   SPUTUM   IN   DISEASE  83 

epithelial  cells  and  varying  numbers  of  red  blood-cor- 
puscles are  found  with  the  microscope. 

6.  Bronchial  Asthma. — The  sputum  during  and  fol- 
lowing an  attack  is  scanty  and  very  tenacious.  Most 
characteristic  is  the  presence  of  Curschmaim's  spirals, 
Charcot-Leyden  crystals,  and  eosinophilic  leukocytes. 

7.  Croupous  Pneumonia. — Characteristic  of  this  dis- 
ease is  a  scanty,  rusty  red,  very  tenacious  sputum,  con- 
taining red  corpuscles  or  altered  blood-pigment,  leuko- 
cytes, epithelial  cells,  usually  many  pneumococci,  and 
often  very  small  fibrinous  casts.  This  sputum  is  seen 
during  the  stage  of  red  hepatization.  During  resolu- 
tion the  sputum  assumes  the  appearance  of  that  of 
chronic  bronchitis.  When  pneumonia  occurs  during  the 
course  of  a  chronic  bronchitis,  the  characteristic  rusty 
red  sputum  may  not  appear. 

8.  Pulmonary  Tuberculosis. — The  sputum  is  varia- 
ble. In  the  earliest  stages  it  may  appear  only  in  the 
morning,  and  is  then  scanty  and  almost  purely  mucoid, 
with  an  occasional  yellow  flake;  or  there  may  be  only 
one  very  small  mucopurulent  mass.  When  the  quantity 
is  small,  there  may  be  no  cough,  the  sputum  reaching 
the  larynx  by  action  of  the  bronchial  cilia.  This  is  not 
well  enough  recognized  by  practitioners.  A  careful  in- 
spection of  all  the  sputum  brought  up  by  the  patient 
on  several  successive  days,  and  a  microscopic  examina- 
tion of  all  yellow  portions,  will  not  infrequently  establish 
a  diagnosis  of  tuberculosis  when  physical  signs  are  nega- 
tive. Intelligent  cooperation  of  the  patient  is  essential 
in  such  cases.  Tubercle  bacilli  will  sometimes  be  found 
in  large  numbers  at  this  stage.  Blood-streaked  sputum 
is  strongly  suggestive  of  tuberculosis,  and  is  more  com- 


84  THE   SPUTUM 

mon  in  the  early  stages  than  later.     It  usually  indicates 
an  advancing  process. 

The  sputum  of  more  advanced  cases  resembles  that  of 
chronic  bronchitis,  with  the  addition  of  tubercle  bacilli 
and  elastic  fibers.  Nummular  masses — circular,  "coin- 
like" disks,  which  sink  in  water — may  be  seen.  Caseous 
particles  containing  immense  numbers  of  the  bacilU  are 
common.  Far-advanced  cases  with  old  cavities  often 
show  rather  firm,  spheric  or  ovoid  grayish  masses  in  a 
thin  fluid — the  so-called  "globular  sputum."  These 
globular  masses  usually  contain  many  tubercle  bacilli. 
Considerable  hemorrhages  are  not  infrequent,  and  for 
some  time  thereafter  the  sputum  may  contain  clots  of 
blood  or  be  colored  brown. 


CHAPTER  II 

THE  URINE 

Preliminary  Considerations. — The  urine  is  an  ex- 
tremely complex  aqueous  solution  of  various  organic 
and  inorganic  substances.  Most  of  the  substances  are 
either  waste-products  from  the  body  metabolism  or 
products  derived  directly  from  the  foods  eaten.  Nor- 
mally, the  total  amount  of  solid  constituents  carried  off 
in  twenty-four  hours  is  about  60  gm.,  of  which  the  organic 
substances  make  up  about  35  gm.  and  the  inorganic  about 
25  gm. 

The  most  important  organic  constituents  are  urea, 
uric  acid,  and  ammonia.  Urea  constitutes  about  one- 
half  of  all  the  soHds,  or  about  30  gm,  in  twenty-four 
hours. 

The  chief  inorganic  constituents  are  the  chlorids, 
phosphates,  and  sulphates.  The  chlorids,  practically 
all  in  the  form  of  sodium  chlorid,  make  up  about  one-half 
of  the  inorganic  substances,  or  about  13  gm.,  in  twenty- 
four  hours. 

Certain  substances  appear  in  the  urine  only  in  patho- 
logic conditions.  The  most  important  of  these  are  pro- 
teins, sugars,  acetone,  and  related  substances,  bile, 
hemoglobin,  and  the  diazo  substances. 
-  In  addition  to  the  substances  in  solution  all  urines 
contain  various  microscopic  structures. 

85 


86  THE   URINE 

While,  under  ordinary  conditions,  the  composition  of 
urine  does  not  vary  much  from  day  to  day,  it  varies 
greatly  at  different  hours  of  the  same  day.  It  is  evident, 
therefore,  that  the  collection  of  the  specimen  is  import- 
ant and  that  no  quantitative  test  can  be  of  value  unless  a 
sample  of  the  mixed  twenty-four-hour  urine  he  used.  The 
patient  should  be  instructed  to  void  all  the  urine  during 
the  twenty-four  fours  into  a  clean  vessel  kept  in  a  cool 
place,  to  mix  it  well,  to  measure  the  whole  quantity,  and 
to  bring  8  or  more  ounces  for  examination.  A  pint 
fruit-jar  is  a  convenient  container.  When  it  is  desired 
to  make  only  qualitative  tests,  as  for  albumin  or  sugar,  a 
"sample"  voided  at  random  will  answer.  It  should  be 
remembered,  however,  that  urine  passed  about  three 
hours  after  a  meal  is  most  likely  to  contain  pathologic 
substances.  That  voided  first  in  the  morning  is  least 
likely  to  contain  them.  To  diagnose  cyclic  albuminuria 
samples  obtained  at  various  periods  during  the  twenty- 
four  hours  must  be  examined. 

The  urine  must  be  examined  while  fresh  Decom- 
position sets  in  rapidly,  especially  in  warm  weather,  and 
greatly  interferes  with  all  the  examinations.  Decom- 
position may  be  delayed  by  adding  5  grains  of  boric 
acid  (as  much  of  the  powder  as  can  be  heaped  upon  a 
ten-cent  piece)  for  each  4  ounces  of  urine.  Formalin, 
in  proportion  of  i  drop  to  4  ounces,  is  also  an  eflSi- 
cient  preservative,  but  if  larger  amounts  be  used,  it  may 
give  reactions  for  sugar  and  albumin,  and  is  likely  to 
cause  a  precipitate  which  greatly  interferes  with  the 
microscopic  examination.  Thymol,  toluol,  and  chloro- 
form are  Hkewise  much  used.  The  chief  objection  to 
toluol  is  the  fact  that  it  floats  upon  the  surface,  and  the 


THE    URINE  87 

urine  must  be  pipeted  from  beneath  it.  Chloroform  is 
probably  the  least  satisfactory.  It  reduces  Fehling's 
solution;  and  it  settles  to  the  bottom  in  the  form  of 
globules  which  it  is  impossible  to  avoid  when  remov- 
ing the  sediment  for  microscopic  examination.  One 
of  these  preservatives  may  be  placed  in  the  vessel 
when  collection  of  the  twenty-four-hour  sample  is 
begun.  Whenever  possible  the  urine  should  be  kept 
on  ice. 

Normal  and  abnormal  pigments,  which  interfere  with 
certain  of  the  tests,  can  be  removed  by  filtering  the  urine 
through  animal  charcoal,  or  precipitating  with  a  solu- 
tion of  acetate  of  lead  and  filtering. 

Certain  cloudy  urines  cannot  be  clarified  by  ordinary 
filtration  through  paper,  particularly  when  the'  cloudi- 
ness is  due  to  bacteria.  Such  urines  can  usually  be 
rendered  perfectly  clear  by  adding  a  small  amount  of 
purified  talc  or  infusorial  earth  (Keiselguhr) ,  shaking 
well,  and  filtering. 

A  suspected  fluid  can  be  identified  as  urine  by  detect- 
ing any  considerable  quantity  of  urea  in  it  (see  p.  114). 
Traces  of  urea  may,  however,  be  met  with  in  ovarian 
cyst  fluid,  while  urine  from  very  old  cases  of  hydrone- 
phrosis may  contain  little  or  none. 

The  frequency  of  micturition  is  often  suggestive  in 
diagnosis.  Whether  it  is  unduly  frequent  can  best  be 
ascertained  by  asking  the  patient  whether  he  has  to  get 
up  at  night  to  urinate.  Increased  frequency  may  be 
due  to  restlessness;  to  increased  quantity  of  urine;  to 
irritability  of  the  bladder,  usually  an  evidence  of  cys- 
titis; to  obstruction  ("retention  with  overflow");  or  to 
paralysis  of  the  sphincter. 


88  THE   URINE 

Clinical  examination  of  the  urine  may  conveniently 
be  considered  under  four  heads:  I.  Physical  examina- 
tion. II.  Chemic  examination.  III.  Microscipic  ex- 
amination.    rV.  The  urine  in  disease. 

L  PHYSICAL  EXAMINATION 

1.  Quantity. — The  quantity  passed  in  twenty-four 
hours  varies  greatly  with  the  amount  of  liquids  ingested, 
perspiration,  etc.  The  normal  may  be  taken  as  looo  to 
15CX)  c.c,  or  35  to  50  ounces  for  an  adult  in  this  country. 
German  writers  give  higher  figures.  For  children  the 
amount  is  somewhat  greater  in  proportion  to  body 
weight. 

The  quantity  is  increased  (polyuria)  during  absorption 
of  large  serous  effusions  and  in  many  nervous  conditions. 
It  is  usually  much  increased  in  chronic  interstitial  nephri- 
tis, diabetes  insipidus,  and  diabetes  mellitus.  In  these 
conditions  a  permanent  increase  in  amount  of  urine  is 
fairly  constant — a  fact  of  much  value  in  diagnosis.  In 
diabetes  mellitus  the  urine  may,  though  rarely,  reach  the 
enormous  amount  of  50  liters. 

The  quantity  is  decreased  (oUguria)  in  severe  diarrhea; 
in  fevers;  in  all  conditions  which  interfere  with  circula- 
tion in  the  kidney,  as  poorly  compensated  heart  disease; 
in  the  parenchymatous  forms  of  nephritis,  and  during 
accumulation  of  fluid  in  the  serous  cavities.  In  uremia 
the  urine  is  usually  very  greatly  decreased  and  may  be 
entirely  suppressed  (anuria). 

Ordinarily,  more  urine  is  voided  during  the  day  than 
during  the  night,  the  normal  ratio  being  about  100  to  50 
or  60.  In  certain  diseases,  notably  arteriosclerosis  and 
cardiac  and  renal  disease,  conditions  are  reversed,  and 


PHYSICAL   EXAMINATION  89 

the  night  urine  (7  p.  m.  to  7  a.  m.)  equals  or  exceeds  that 
passed  during  the  day. 

2.  Color. — This  varies  considerably  in  health,  and 
depends  largely  upon  the  quantity  of  urine  voided,  dilute 
urines  being  pale  and  concentrated  urines  highly  colored. 
The  usual  color  is  yellow  or  reddish  yellow,  due  to  the 
presence  of  several  pigments,  chiefly  urochrome,  which 
is  yellow.  Traces  of  hematoporphyrin,  uroerythrin, 
and  urobilin  are  frequent.  Uroerythrin  is  chiefly  re- 
sponsible for  the  deep  reddish  tinge  of  urine  in  acute 
fevers.  Urobilin  has  cUnical  significance  and  is  dis- 
cussed later  (see  p.  158).  Acid  urine  is  generally  darker 
than  alkaline.  In  recording  the  color,  Vogel's  scale  (see 
Plate  III)  is  very  widely  used,  the  urine  being  filtered 
and  examined  by  transmitted  Hght  in  a  glass  3  or  4 
inches  in  diameter. 

Color  is  sometimes  greatly  changed  by  abnormal 
pigments.  Blood-pigment  gives  a  red  or  brown,  smoky 
color.  Urine  containing  bile  is  yellowish  or  brown,  with 
a  yellow  foam  when  shaken.  It  may  assume  a  greenish 
hue  after  standing,  owing  to  oxidation  of  bilirubin  into 
biliverdin.  Ingestion  of  small  amounts  of  methylene 
blue  gives  a  pale  green;  large  amounts  give  a  marked 
greenish  blue.  Santonin  produces  a  yellow;  rhubarb, 
senna,  cascara,  and  some  other  cathartics,  a  brown 
color;  these  change  to  red  upon  addition  of  an  alkaK, 
and  if  the  urine  be  alkaline  when  voided,  may  cause 
suspicion  of  hematuria.  A  bright  pink  or  red  color 
appearing  when  the  urine  is  alkaHnized  may  be  due  to 
phenolphthalein.  Thymol  gives  a  yellowish  green. 
Following  poisoning  from  phenol  and  related  drugs  the 
urine  may  have  a  normal  color  when  voided,  but  becomes 


90  THE   URINE 

olive  green  to  brownish  black  upon  standing.  In  sus- 
ceptible individuals  therapeutic  doses  of  creosote,  or 
absorption  from  carbolized  dressings,  may  cause  this 
change.  Urine  which  contains  melanin,  as  sometimes 
in  melanotic  sarcoma,  and  very  rarely  in  wasting  dis- 
eases, also  becomes  brown  or  black  upon  long  standing. 
A  similar  darkening  upon  exposure  to  the  air  occurs 
in  alkaptonuria  (see  p.  157).  A  milky  color  may  be  due 
to  presence  of  chyle,  or  milk  may  have  been  added  by 
a  malingering  patient. 

A  pale  greenish  urine  with  high  specific  gravity 
strongly  suggests  diabetes  mellitus. 

3.  Transparency. — Freshly  passed  normal  urine  is 
clear.  Upon  standing,  a  faint  cloud  of  mucus,  leuko- 
cytes, and  epitheUal  cells  settles  to  the  bottom — the  so- 
called  "nubecula."  This  is  more  abundant  in  women 
owing  to  vaginal  cells  and  mucus.  In  urines  of  high 
specific  gravity  it  may  float  near  the  middle  of  the  fluid. 

Abnormal  cloudiness  is  usually  due  to  presence  of 
phosphates,  urates,  pus,  blood,  or  bacteria. 

Amorphous  phosphates  are  precipitated  in  neutral  or 
alkaHne  urine.  They  form  a  white  cloud  and  sediment, 
which  disappear  upon  addition  of  an  acid. 

Amorphous  urates  are  precipitated  only  in  acid  urine. 
They  form  a  white  or  pink  cloud  and  sediment  ("brick- 
dust  deposit"),  which  disappear  upon  heating. 

Pus  resembles  amorphous  phosphates  to  the  naked 
eye.  Its  nature  is  easily  recognized  with  the  microscope, 
or  by  adding  a  strong  solution  of  caustic  soda  to  the 
sediment,  which  is  thereby  transformed  into  a  gelatinous 
mass  (Donne's  test). 

Blood  gives  a  reddish  or  brown,  smoky  color,  and  may 


PLATE  III 


m 


PALE  YELLOW. 


LIGHT  YELLOW. 


III. 
YELLOW. 


IV. 

REDDISH  YELLOW. 


V. 

YELLOWISH  RED, 


VI. 

RED. 


VII. 

BROWNISH  RED. 


VIII. 

REDDISH  BROWN 


BROWNISH  BLACK. 


Scale  of  Urinary  Colors,  according  to  Vogel 


PHYSICAL   EXAMINATION  9I 

be  recognized  with  the  microscope  or  by  tests  for  hemo- 
globin. 

Bacteria,  when  present  in  great  numbers,  give  a  uni- 
form cloud,  which  cannot  be  removed  by  ordinary  filtra- 
tion.   They  are  detected  with  the  microscope.     "^ 

The  cloudiness  of  decomposing  urine  is  due  mainly  to 
precipitation  of  phosphates  and  multipUcation  of  bac- 
teria. 

4.  Odor. — The  characteristic  aromatic  odor  is  due  to 
volatile  acids,  and  is  most  marked  in  concentrated  urines. 
During  decomposition  the  odor  becomes  ammoniacal. 
A  fruity  odor  is  sometimes  noted  in  diabetes,  due  prob- 
ably to  acetone.  Urine  which  contains  cystin  may  de- 
velop an  odor  of  sulphureted  hydrogen  during  decom- 
position. 

Various  articles  of  diet  and  drugs  impart  peculiar 
odors.  Notable  among  these  are  asparagus,  which  gives 
a  characteristic  offensive  odor,  and  turpentine,  which 
imparts  an  odor  somewhat  suggesting  that  of  violets. 

5.  Reaction. — Normally,  the  mixed  twenty-four-hour 
urine  is  slightly  acid  in  reaction.  The  acidity  sometimes 
increases  for  a  time  after  the  urine  is  voided,  the  so- 
called  "acid  fermentation."  The  acidity  was  formerly 
held  to  be  due  wholly  to  acid  phosphates,  but  Folin 
has  shown  that  the  acidity  of  a  clear  urine  is  ordinarily 
much  greater  than  the  acidity  of  all  the  phosphates,  the 
excess  being  due  to  free  organic  acids.  Individual 
samples  may  be  slightly  alkaline,  especially  after  a  full 
meal;  or  they  may  be  amphoteric,  turning  red  litmus- 
paper  blue  and  blue  paper  red,  owing  to  presence  of  both 
alkaline  and  acid  phosphates.  The  reaction  is  deter- 
mined  by  means  of   litmus-paper,  which,   however,   is 


93  THE  X7RINE 

worthless  unless  of  good  quality.    That  put  up  in  vials 
by  Squibb  can  be  recommended. 

Acidity  is  increased  after  administration  of  certain 
drugs,  and  whenever  the  urine  is  concentrated  from  any 
cause,  as  in  fevers.  A  strongly  acid  urine  may  cause  fre- 
quent micturition  because  of  its  irritation.  This  is  often 
an  important  factor  in  the  troublesome  enuresis  of 
children. 

The  urine  always  becomes  alkaline  upon  long  standing, 
owing  to  decomposition  of  urea  with  formation  of  am- 
monia. If  markedly  alkaline  when  voided,  it  usually 
indicates  such  "ammoniacal  decomposition"  in  the 
bladder,  which  is  the  rule  in  chronic  cystitis,  especially 
that  due  to  paralysis  or  obstruction.  Alkalinity  due  to 
anmionia  (volatile  alkalinity)  can  be  distinguished  by  the 
fact  that  Utmus-paper  turned  blue  by  the  urine  again 
becomes  red  upon  gentle  heating,  or  that  the  paper  will 
turn  blue  when  held  in  the  steam  over  the  boiling  urine. 
A  second  form  of  alkalinity,  fixed  alkalinity,  is  due  to 
alkaline  salts,  and  is  often  observed  during  frequent 
vomiting,  after  the  crisis  of  pneumonia,  in  various  forms 
of  anemia,  after  full  meals,  and  after  administration  of 
certain  drugs,  especially  salts  of  vegetable  acids 
-  Quantitative  estimation  of  acidity  of  urine  is  not  of 
much  clinical  value.  When,  however,  it  is  desired  to 
make  it,  the  method  of  Folin  will  be  foimd  satisfactory. 
In  every  case  the  sample  must  be  from  the  mixed  twenty- 
four-hour  urine  and  as  fresh  as  possible. 

Folin's  Method. — Into  a  small  flask  measure  25  c.c.  of 
the  urine  and  add  i  or  2  drops  0.5  per  cent,  alcoholic  solu- 
tion of  phenolphthalein  and  15  or  20  gm.  of  neutral  potassium 


PHYSICAL   EXAMINATION  93 

oxalate.  Shake  for  a  minute,  and  immediately  titrate  with 
decinormal  sodium  hydroxid,  shaking  meanwhile,  until  the' 
first  permanent  pink  appears.  Read  oflf  from  the  buret  the 
amount  of  decinormal  sodium  hydroxid  solution  added,  and 
calculate  the  number  of  cubic  centimeters  which  would  be 
required  for  the  entire  twenty-four  hours'  urine.  Folin 
places  the  normal  acidity,  obtained  in  this  way,  at  617. 

6.  Specific  Gravity. — The  normal  average  is  about 
1. 01 7  to  1,020.  Samples  of  urine  taken  at  random  may 
go  far  above  or  below  these  figures,  hence  a  sample  of  the 
mixed  twenty-four-hour  urine  should  always  be  used. 

Pathologically,  it  may  vary  from  i.ooi  to  1.060.  It 
is  low  in  chronic  interstitial  nephritis,  diabetes  insipidus, 
and  many  functional  nervous  disorders.  It  is  high  in 
fevers  and  in  parenchymatous  forms  of  nephritis.  In 
any  form  of  nephritis  a  sudden  fall  without  a  corre- 
sponding increase  in  quantity  of  urine  may  foretell  ap- 
proaching uremia.  It  is  highest  in  diabetes  mellitus.  A 
high  specific  gravity  when  the  urine  is  not  highly  colored 
should  lead  one  to  suspect  this  disease.  A  normal 
specific  gravity  does  not,  however,  exclude  it. 

The  specific  gravity  is  most  conveniently  estimated  by 
means  of  the  urinometer  (Fig.  17).  Squibb's  urinometer 
is  adjusted  to  give  accurate  readings  at  22.5°  C;  most 
other  instruments,  at  15°  C.  If  the  urine  be  brought  to 
about  the  right  temperature,  a  correction  for  tempera- 
ture will  seldom  be  necessary  in  cHnical  work.  For 
accuracy,  however,  it  is  necessary  to  add  o.ooi  to  the 
urinometer  reading  for  each  3°  C.  above  the  tempera- 
ture for  which  the  urinometer  is  standardized,  and  to 
subtract  o.ooi  for  each  3°  C.  below  that  point.  Care 
should  be  taken  that  the  urinometer  does  not  touch  the 


94 


THE   URINE 


side  of  the  tube,  and  that  air-bubbles  are  removed  from 
the  surface  of  the  urine.  Bubbles  are  easily  removed 
with  a  strip  of  filter-paper.  With  most  instruments  the 
reading  is  taken  from  the  bottom  of  the  meniscus.  A 
long  scale  on  the  stem  is  desirable,  because  of  the  greater 
ease  of  accurate  reading.  Many  of  the  urinometers  on 
the  market  are  too  small  to  be  of  any  real  value. 

One  frequently  wishes  to  ascertain  the  specific  gravity 
of  quantities  of  fluid  too  small  to  float   a  urinometer. 


Fig.  17. — Squibb's  urinometer  with  thennometer  and  cylinder. 


A  simple  device  for  this  purpose,  which  requires  only 
about  3  c.c.  and  is  very  satisfactory  in  clinical  work,  has 
been  designed  by  Saxe  (Fig.  18).  The  urine  is  placed  in 
the  bulb  at  the  bottom,  the  instrument  is  floated  in  dis- 
tilled water,  and  the  specific  gravity  is  read  off  from  the 
scale  upon  the  stem. 

7.  Total  Solids. — An  estimation  of  the  total  amount 
of  soUds  which  pass  through  the  kidneys  in  twenty-four 
hours  is,  in  practice,  one  of  the  most  useful  of  urinary 
examinations.    The  normal  for  a  man  of  150  pounds  is 


PHYSICAL    EXAMINATION 


95 


about  60  gm,,  or  950  gr.  The  principal  factors  which 
influence  this  amount  are  body  weight  (except  with 
excessive  fat),  diet,  exercise,  and  age,  and  these  should 
be  considered  in  making  an  estimation.  After  about 
the  forty-fifth  year  it  becomes  gradually  less;  after 
seventy-five  years  it  is  about  one-half  the  amount  given. 


Fig.  18. — Saxe's  urinopyknometer  and  jar  for  same. 


In  disease,  the  amount  of  solids  depends  mainly  upon 
the  activity  of  metaboHsm  and  the  abiHty  of  the  kidneys 
to  excrete.  An  estimation  of  the  solids,  therefore, 
furnishes  an  important  clue  to  the  functional  eflSciency 
of  the  kidneys.  The  kidneys  bear  much  the  same 
relation  to  the  organism  as  does  the  heart:  they  cause 
no  direct  harm  so  long  as  they  are  capable  of  perform- 


96  THE  URINE 

ing  the  work  required  of  them.  When,  however,  through 
either  organic  disease  or  functional  inactivity,  they  fail 
to  carry  ofiF  their  proportion  of  the  waste-products  of  the 
body,  some  of  these  products  must  either  be  eliminated 
through  other  organs,  where  they  cause  irritation  and 
disease,  or  be  retained  within  the  body,  where  they  act 
as  poisons.  The  great  importance  of  these  poisons  in 
production  of  distressing  symptoms  and  even  organic 
disease  is  not  well  enough  recognized  by  most  practi- 
tioners. Disappearance  of  unpleasant  and  perplexing 
symptoms  as  the  urinary  soUds  rise  to  the  normal  under 
proper  treatment  is  often  most  surprising. 

When,  other  factors  remaining  unchanged,  the  amount 
of  solids  eliminated  is  considerably  above  the  normal, 
increased  destructive  metabolism  may  be  inferred. 

The  total  solids  can  be  estimated  roughly,  but  ac- 
curately enough  for  most  cHnical  purposes,  by  multi- 
plying the  last  two  figures  of  the  specific  gravity  of  the 
mixed  twenty-four-hour  urine  by  the  number  of  ounces 
voided  and  to  the  product  adding  one-tenth  of  itself. 
This  gives  the  amount  in  grains.  If,  for  example,  the 
twenty-four-hour  quantity  is  3  pints  or  48  ounces,  and 
the  specific  gravity  is  1.018,  the  total  solids  would  ap- 
proximate 950  gr.,  as  follows: 

48  X  18  =  864;  864  +  86.4  =  950.4. 

This  method  is  especially  convenient  for  the  practi- 
tioner, because  patients  nearly  always  report  the  amount 
of  urine  in  pints  and  ounces,  and  it  avoids  the  necessity 
of  converting  into  the  metric  system.  Haser's  method, 
which  uses  the  metric  system,  is  more  widely  used,  but 
is  less  convenient.    The  last  two  figures  of  the  specific 


PHYSICAL   EXAMINATION  97 

gravity  are  multiplied  by  2.33.  The  product  is  then  mul- 
tiplied by  the  number  of  cubic  centimeters  voided  in 
twenty-four  hours  and  divided  by  icxx3.  This  gives  the 
total  soHds  in  grams. 

8.  Functional  Tests. — Within  the  past  few  years 
much  thought  has  been  devoted  to  methods  of  more 
accurately  ascertaining  the  functional  efficiency  of  the 
kidneys,  especially  of  one  kidney  when  removal  of  the 
other  is  under  consideration.  The  most  promising  of 
the  methods  which  have  been  devised  are  cryoscopy, 
electric  conductivity,  the  phloridzin  test,  the  methylene- 
blue  test,  and  the  phenolsulphonephthalein  test.  It  is 
doubtful  whether,  except  in  the  case  of  the  last,  these 
yield  any  more  information  than  can  be  had  from  an 
intelHgent  consideration  of  the  specific  gravity  and  the 
twenty-four-hour  quantity,  together  with  a  microscopic 
examination.  They  are  most  useful  when  the  urines 
obtained  from  separate  kidneys  by  segregation  or  ure- 
teral catheterization  are  compared.  Only  the  phenol- 
sulphonephthalein test  will  be  given  in  detail.  The 
reader  is  referred  to  larger  works  upon  urinalysis  for 
details  regarding  the  others. 

Cryoscopy,  determination  of  the  freezing-point,  de- 
pends upon  the  principle  that  the  freezing-point  of  a 
fluid  is  depressed  in  proportion  to  the  number  of  mole- 
cules, organic  and  inorganic,  in  solution.  To  have  any 
value,  the  freezing-point  of  the  urine  must  be  compared 
with  that  of  the  blood,  since  it  is  not  so  much  the  num- 
ber of  molecules  contained  in  the  urine,  as  the  number 
which  the  kidney  has  failed  to  carry  off  and  has  left  in 
the  blood,  that  indicates  its  insufficiency. 

Electric  conductivity  refers  to  the  power  of  the  urine 

7 


98  THE   URINE 

to  carry  an  electric  current.  It  is  increased  in  pro- 
portion to  the  number  of  inorganic  molecules  in  solution. 

In  the  methylene-blue  test  of  Achard  and  Castaigne 
a  solution  of  methylene  blue  is  injected  intramuscularly, 
and  the  time  of  its  appearance  in  the  urine  is  noted. 
Normally,  it  appears  in  about  thirty  minutes.  When 
delayed,  renal  "permeability"  is  supposed  to  be  inter- 
fered with.  Since  methylene  blue  is  sometimes  ex- 
creted as  a  colorless  derivative,  indigo-carmin  has  been 
proposed  as  a  substitute.  In  the  absence  of  renal  in- 
sufficiency this  always  gives  a  blue  color,  which  begins 
to  appear  in  about  five  minutes. 

The  phloridzin  test  consists  in  the  hypodermic  injec- 
tion of  a  small  quantity  of  phloridzin.  This  substance 
is  transformed  into  glucose  by  the  kidneys  of  healthy 
persons.  In  disease,  this  change  is  more  or  less  inter- 
fered with,  and  the  amount  of  glucose  recoverable  from 
the  urine  is  taken  as  an  index  of  the  secretory  power  of 
the  kidneys. 

Phenolsulphonephthalein  Test. — This  test,  which  was 
otTered  by  Rowntree  and  Geraghty  in  1910,  consists  in 
the  intramuscular  (or  intravenous)  injection  of  a  solu- 
tion of  phenolsulphonephthalein,  a  drug  which  is  elimi- 
nated only  by  the  kidneys,  and  whose  amount  in  the 
urine  is  easily  estimated  by  colorimetric  methods.  The 
time  of  its  first  appearance  in  the  urine  and  the  quantity 
eliminated  within  a  definite  period  are  taken  as  a  measure 
of  the  functional  capacity  of  the  kidneys.  The  test  is 
harmless,  comparatively  simple,  and  apparently  reliable. 
It  will  sometimes  reveal  a  very  serious  degree  of  renal 
failure  when  total  quantity,  total  solids,  and  urea  are 
practically  normal. 


PHYSICAL   EXAMINATION  99 

Preparation  of  Solution. — The  solution  used  consists  of — 

Phenolsulphonephthalein 0.6    gm.; 

jj  Sodium  hydroxid 0.84  c.c; 

0.75  per  cent,  sodium  chlorid  to 100    " 

To  this  is  added  just  enough  of  the  ^  sodium  hydroxid  solu- 
tion to  change  the  color  to  a  beautiful  Bordeaux  red,  usually 
2  to  3  drops.    An  approximate  ^  solution  of  sodium  hydroxid 

can  be  made  by  dissolving  82  gm.  of  the  chemically  pure 
alkali  in  a  liter  of  distilled  water.  The  phenolsulphonephthal- 
ein solution  can  be  purchased  in  ampoules  ready  for  use. 

Technic. — Give  the  patient  300  to  400  c.c.  of  water  to 
promote  secretion  of  urine.  Twenty  to  thirty  minutes  after- 
ward empty  the  bladder  with  a  catheter.  Leaving  the  cath- 
ter  in  place,  inject  i  c.c.  of  the  above  solution  (previously 
sterilized  by  boiling)  into  the  lumbar  muscles  with  a  hypoder- 
mic syringe.  Allow  the  urine  to  drain  into  a  test-tube  in 
which  I  drop  of  25  per  cent,  sodium  hydroxid  has  been  placed, 
and  note  the  time  of  the  first  appearance  of  a  faint  but  dis- 
tinct pink  color.  Then,  unless  there  be  prostatic  obstruction, 
withdraw  the  catheter  and  instruct  the  patient  to  urinate  at 
the  end  of  an  hour  from  the  first  appearance  of  the  color, 
and  again  at  the  end  of  the  second  hour,  saving  the  urine  in 
separate  vessels.  If  there  be  objection  to  catheterizing,  the 
patient  may  empty  his  bladder  voluntarily  when  the  drug 
is  injected  and  then  urinate  every  few  minutes  until  the  pink 
color  appears. 

Since  the  principal  interest  is  not  in  the  time  of  first  ap- 
pearance, but  in  the  quantity  eliminated,  the  test  may  be 
simplified  by  having  the  patient  empty  his  bladder  when  the 
drug  is  injected,  and  again  one  hour  and  ten  minutes  and 
two  hours  and  ten  minutes  afterward. 

Dilute  each  of  the  two  portions  to  exactly  1000  c.c.  with 


lOO  THE  URINE 

water.  This  is  best  done  in  i -liter  volumetric  flasks.  The 
color  is  yellow  or  orange  while  the  urine  is  acid,  but  becomes 
brilliant  purple  red  when  it  is  alkalinized.  Add  sufl&cient 
25  per  cent,  sodium  hydroxid  solution  to  bring  out  the  maxi- 
mum color,  filter  off  a  small  quantity,  and  estimate  the  amount 
of  phenolsulphonephthalein  excreted  during  each  of  the 
one-hour  periods,  by  comparing  its  color  with  that  of  a  stand- 
ard alkaline  solution  containing  0.006  gm.  of  phenolsul- 
phonephthalein (i  c.c.  of  original  solution)  to  the  liter.  This 
can  be  done  with  a  fair  degree  of  accuracy  in  50-c.c.  cylinders, 
the  darker  fluid  being  diluted  until  the  two  have  exactly 
the  same  depth  of  color.  They  then  contain  the  same 
percentage  of  the  drug;  and,  the  amount  in  the  standard 
solution  being  known,  it  is  easy  to  calculate  the  amount  in 
the  total  quantity  of  urine.  The  cylinders  may  be  viewed 
from  the  top,  or  they  may  be  compared  in  a  frame  with  a 
ground  glass  back  like  that  of  the  Sahli  hemoglobinometer. 
For  greater  acciu-acy  the  Rowntree  and  Geraghty  modifica- 
tion of  the  Hellige  colorimeter  is  recommended.  When  it 
is  necessary  to  defer  the  color  comparison  for  hours  or  days 
the  urine  must  be  kept  acid,  as  the  color  generally  fades 
after  it  is  alkalinized. 

Normal  Standards. — Under  normal  conditions  the  drug 
first  appears  in  the  urine  in  five  to  eleven  minutes-  after  the 
injection.  Within  the  first  hour  after  its  appearance  40  to 
60  per  cent,  is  eliminated;  in  the  two  hours,  60  to  85  per  cent. 
Pathologically  the  elimination  may  be  reduced  to  a  trace 
or  even  none  at  all  in  the  two  hours. 

n.  CHEMIC  EXAMINATION 
A.   Normal  Constituents 
Of  the  large  number  of  organic  and  inorganic  sub- 
stances normally  present  in  the  urine,  only  a  few  demand 
any  consideration  from  the  clinician.     The   following 


CHEMIC   EXAMINATION  lOI 

table,  therefore,  outlines  the  average  composition  from 
the  clinical,  rather  than  from  the  chemical,  standpoint. 
Only  the  twenty-four-hour  quantities  are  given,  since 
they  alone  furnish  an  accurate  basis  for  comparison. 
The  student  cannot  too  soon  learn  that  percentages  mean 
little  or  nothing,  excepting  as  they  furnish  a  means  of 
calculating  the  twenty-four-hour  elimination. 

COMPOSITION  OF  NORMAL  URINE 

Grams  in  twenty-  Approximate 
four  hours.  average. 

Water looo— 1500          1200 

Total  substances  in  solution S5-70                 60 

Inorganic  substances 20-30                  25 

Chlorids  (chiefly  sodium  chlorid) 10-15                   12.5 

Phosphates  (estimated  as  phosphoric  acid), 

total 2.5-3.5                  3 

Earthy,  5  of  total i 

Alkaline,  f  of  total 2 

Sulphates   (estimated  as  sulphuric  acid), 

total I-5-3-0                   2.5 

Mineral,  ^^  of  total 2.25 

Conjugate,  ^^  of  total 0.25 

Includes  indican Trace 

Ammonia 0.5-1.0                  0.7 

Organic  substances 30-40                  35 

Urea 30-35                 30 

Uric  acid 0.4-1.0                  0.7 

Although  the  conjugate  sulphates  are  organic  com- 
pounds, they  are,  for  the  sake  of  convenience,  included 
with  the  inorganic  sulphates  in  the  above  table. 

Among  constituents  which  are  of  little  clinical  im- 
portance, or  are  present  only  in  traces,  are: 

Inorganic. — Iron,  carbonates,  nitrates,  silicates,  and 
fluorids. 

Organic. — Creatinin,  hippuric  acid,  PWUi  bases,  oxalic 

CULLLlit  UK  Ui^TLUl-'/ni 


102 


THE   URINE 


Fig.  ig. — Graphic  expression  of  quantities  in  the  urine.    Solid  line,  normal  urine;  dotted 
line,  an  example  of  pathologic  urine  in  a  case  of  cancerous  cachexia  (Saxej. 

']  I.  /if3  )^ 


CHEMIC   EXAMINATION  IO3 

acid,  benzoic  acid,  volatile  fatty  acids,  pigments,  and 
acetone. 

Variations  in  body  weight,  diet,  and  exercise  cause 
marked  fluctuations  in  the  total  solids  and  in  individual 
substances. 

1.  Chlorids. — These  are  derived  from  the  food,  and 
are  mainly  in  the  form  of  sodium  chlorid.  The  amount 
excreted  normally  is  10  to  15  gm.  in  twenty-four  hours. 
It  is  much  affected  by  the  diet,  and  is  reduced  to  a  mini- 
mum in  starvation. 

Excretion  of  chlorids  is  diminished  in  nephritis  and 
in  fevers,  especially  in  pneumonia  and  inflammations 
leading  to  the  formation  of  large  exudates.  In  nephritis 
the  kidneys  are  less  permeable  to  the  chlorids,  and  it  is 
possible  that  the  edema  is  due  largely  to  an  effort  of 
the  body  to  dilute  the  chlorids  which  have  been  retained. 
Certainly  an  excess  of  chlorids  in  the  food  will  increase 
both  the  albuminuria  and  the  edema  of  nephritis.  In 
fevers  the  diminution  is  due  largely  to  decrease  of  food, 
though  probably  in  some  measure  to  impaired  renal 
function.  In  pneumonia  chlorids  are  constantly  very 
low,  and  in  some  cases  are  absent  entirely.  Following 
the  crisis  they  are  increased.  In  inflammations  leading 
to  formation  of  large  exudates — e.  g.,  pleurisy  with 
effusion — chlorids  are  diminished,  because  a  consider- 
able amount  becomes  "locked  up"  in  the  exudate. 
During  absorption  chlorids  are  liberated  and  appear 
in  the  urine  in  excessive  amounts. 

Diminution  of  chlorids  is  also  observed  in  severe 
diarrhea,  anemic  conditions,  and  carcinoma  of  the 
stomach. 

Detection  of  Chlorids. — The  following  simple  test  will 


I04  THE   URINE 

show  the  presence  of  chlorids,  and  at  the  same  time 
roughly  indicate  any  pronounced  alteration  in  amount: 

To  a  few  cubic  centimeters  of  urine  in  a  test-tube  add  a 
few  drops  of  nitric  acid  to  prevent  precipitation  of  phosphates 
and  then  a  few  drops  of  silver  nitrate  solution  of  about  12 
per  cent,  strength.  A  white,  curdy  precipitate  of  silver 
chlorid  forms.  If  the  urine  merely  becomes  milky  or  opales- 
cent, chlorids  are  markedly  diminished. 

Quantitative  Estimation. — The  well-known  and  re- 
Uable  Volhard  method  has  been  simplified  by  Strauss, 
and  this  modification  has  recently  been  still  further 
simphfied  by  Bayne- Jones  and  by  McLean  and  Selling, 
so  that  the  method  is  now  available  for  ordinary  cHn- 
ical  work.  The  only  difficulty  is  the  preparation  of  solu- 
tions, and  these  can  be  purchased  ready  prepared.  A 
less  accurate,  though  simple  and  very  useful,  method  is 
afforded  by  the  centrifuge  (see  p.  105). 

Simplified  Volhard  Method. — Albumin  need  not  be  re- 
moved. In  an  accurately  graduated  50-c.c.  cylinder  place 
5  c.c,  of  the  urine  and  10  c.c.  of  Solution  No.  i.  MLx  by  in- 
verting several  times.  If  a  reddish  color  appears,  add  3  drops 
of  10  per  cent,  potassium  permanganate.  After  five  minutes 
add  Solution  No.  2,  a  very  little  at  a  time,  mixing  after  each 
addition,  until  a  permanent  red-brown  color  (best  seen  against 
a  white  backgroimd)  appears.     This  is  the  end-point. 

The  solutions  are  so  balanced  that  if  the  urine  be  chlorid- 
free  the  volume  of  fluid  when  the  end-point  is  reached  will 
be  35  c.c,  and  that  for  each  gram  per  liter  of  chlorids  in 
the  urine  the  volume  will  be  i  c.c.  less.  Therefore,  the  dif- 
ference between  35  c.c.  and  the  height  of  the  fluid  at  the 
end  of  the  test  gives  directly  the  number  of  grams  of  chlo- 


CHEMIC   EXAMINATION  105 

rids  per  liter  of  urine,  expressed  as  sodium  chlorid.  If,  for 
example,  the  fluid  reaches  the  28  c.c.  mark,  35  — 28  =  7  gm. 
of  sodium  chlorid  per  liter  of  urine. 

A  certified  50-c.c.  graduated  cylinder,  with  glass  stopper, 
is  required.     The  ordinary  50-c.c.  graduate  is  inaccurate. 

The  solutions  are  as  follows: 

No.  I. — Standard  silver  nitrate  solution: 

Silver  nitrate  (C.  P.,  anhydrous,  crystallized) .  29.055  gm.; 

Nitric  acid  (25  per  cent.) 900  c.c; 

Ammonioferric  alum  (cold  saturated  solution)  50    " 

Distilled  water  to 1000    " 

No.   2. — Ammonium  sulphocyanate  solution: 

Ammonium  sulphocyanate 7  gm.; 

Distilled  water 1000  c.c. 

This  solution  is  intentionally  made  too  strong,  and  it  must 
be  standardized  by  diluting  with  distilled  water  until  exactly 
20  c.c.  (and  no  less)  will  produce  a  red  color  when  mixed 
with  exactly  10  c.c.  of  Solution  No.  i. 

Purdy's  Centrifugal  Methods. — As  shown  by  Purdy, 
the  centrifuge  offers  an  important  means  of  making 
quantitative  estimations  of  a  number  of  substances  in 
the  urine.  Results  are  easily  and  quickly  obtained,  and 
are  probably  accurate  enough  for  most  clinical  purposes. 

In  general,  the  methods  consist  in  precipitating  the 
substance  to  be  estimated  in  a  graduated  centrifuge  tube, 
and  applying  a  definite  amount  of  centrifugal  force  for  a 
definite  length  of  time,  after  which  the  percentage  of 
precipitate  is  read  off  upon  the  side  of  the  tube.  Al- 
bumin, if  present,  must  be  previously  removed  by  boil- 
ing and  filtering.  Results  are  in  terms  of  hulk  of  pre- 
cipitate, which  must  not  be  confused  with  percentage 


io6 


THE   URINE 


Fig.  »o. — The  Purdy  electric  centrifuge  with  four  arras. 


Fig.  21      W.itir-motor  centrifuge. 


by  weight.     The  weight  percentage  can  be  found  by 
referring  to  Purdy's  tables,  given  later.     In  this,  as  in 


CHEMIC   EXAMINATION 


107 


all  quantitative  urine  work,  percentages  mean  little  in 
themselves;  the  actual  amount  eliminated  in  twenty- 
four  hours  should  always  be  calculated. 

The  centrifuge  should  have  an  arm  with  a  radius  of  6f 
inches  when  in  motion,  and  should  be  capable  of  main- 
taining a  speed   of   1500   revolutions  a  minute.     The 


c.c.\ 


i~i 


Ie 


il    I        h 


Fig.  22. — Purdy's  tubes  for  the  centrifuge:  a,  Percentage  tube;  h,  sediment  tube. 


electric  centrifuge  is  best,  although  good  work  can  be 
done  with  a  water-power  centrifuge  or,  after  a  little 
practice,  with  the  hand  centrifuge.  A  speed  indicator 
is  desirable  with  electric  and  water-motor  machines, 
although  one  can  learn  to  estimate  the  speed  by  the 
musical  note. 


io8 


THE   URINE 


TABLE  FOR  THE  ESTIMATION  OF  CHLORIDS  AFTER 
CENTRIFUGATION 

Showing  the  bulk-percentage  of  silver  chlorid  {AgCl)  and  the  correspond- 
ing gravimetric  percentages  and  grains  per  fluidounce  of  sodium  chlorid 
{NaCl)  and  chlorin  {Cl).—{Purdy.)    . 


f 

4 

5 

d 

d 

0 

0 

pq 

d 
« 

1 

1 

0 

d 

s 

d 
6 

5 

i 

0.03 

o.is 

0.02 

0.1 

8 

1.04 

4.98 

0.63 

3.02 

* 

0.07 

0.31 

0.04 

0.19 

8i 

I.I 

529 

0.67 

3.22 

f 

O.I 

0.47 

0.06 

0.28 

9, 

1. 17 

5-6 

0.71 

3-4 

1 

0.13 

0.62 

0.08 

0.38 

9i 

1.23 

5-91 

0-75 

3-6 

ij 

0.16 

0.78 

O.I 

0.48 

10 

1-3 

6.22 

0.79 

3-79 

li 

0.19 

0-93 

0.12 

0-57 

loj 

1.36 

6.53 

0.83 

3-97 

if 

0.23 

1.09 

0.14 

0.67 

II 

1-43 

6.84 

0.87 

4.16 

2 

0.26 

1.24 

0.16 

0.76 

"i 

1.49 

7.2 

0.91 

4-35 

2i 

0.29 

1.41 

0.18 

0.85 

12 

1.56 

7.46 

0-95 

4-54 

2i 

0.32 

1.56 

0.2 

0.96 

12J 

1.62 

7.78 

0.99 

4-73 

2| 

0.36 

1.71 

0.22 

1.04 

'^, 

1.69 

8.09 

1.02 

4.92 

3, 

0-39 

1.87 

0.24 

I-I3 

i3i 

1-75 

8.4 

1.06 

5-II 

3i 

0.42 

2.02 

0.26 

1.23 

14 

1.82 

8.71 

I.I 

5-29 

3t 

0.4S 

2.l8 

0.28 

1.32 

i4i 

1.88 

9.02 

1. 14 

5-49 

3l 

0.49 

2-35 

0-3 

1.42 

15^ 

1.94 

9-33 

1. 18 

5-67 

4 

0.52 

2.49 

.0.32 

I-5I 

iSi 

2.01 

965 

1.22 

5.86 

4i 

0-55 

2.64 

0.34 

1.61 

16 

2.07 

9.94 

1.26 

6.06 

4J 

0.58 

2.8 

0.35 

1-7 

16J 

2.-4 

10.27 

r-3 

6.24 

4l 

0.62 

2.96 

0-37 

1.8 

'7, 

2.L' 

10.51 

1-34 

6.43 

5, 

0.65 

3" 

0-39 

1.89 

i7i 

2.27 

10.87 

1.38 

6.62 

si 

0.71 

342 

0-43 

2.09 

18 

2-33 

II. 2 

1.42 

6.81 

6 

0.78 

3-73 

0.47 

2.27 

18J 

2.4 

11.51 

1.46 

7.0 

6J 

0.84 

4-05 

0.51 

2.46 

'9, 

2.46 

11.82 

1-5 

7.19 

7 

0.91 

4-35 

0-5S 

2.62 

195 

2-53 

12.13 

1.54 

7-38 

7J 

0.97 

4.67 

0-59 

2.84 

20 

2-59 

12.44 

1.58 

756 

Bulk-percentage  to  be  read  on  the  side  of  the  tube. 


Estimation  of  Chlorids. — Fill  the  graduated  tube  to  the 
10  c.c.  mark  with  urine;  add  15  drops  strong  nitric  acid  and 
then  silver  nitrate  solution  of  12  per  cent,  strength  to  the 
15  c.c.  mark.  Mix  by  inverting  several  times.  Let  stand  a 
few  minutes  for  a  precipitate  to  form,  and  then  revolve  in  the 


CHEMIC   EXAMINATION  lOQ 

centrifuge  for  three  minutes  at  1200  revolutions  a  minute. 
Each  one-tenth  cubic  centimeter  of  precipitate  equals  i  per 
cent,  by  bulk.  This  may  be  converted  into  percentage  by 
weight  of  chlorin  or  sodium  chlorid  by  means  of  the  table 
upon  page  108. 

2.  Phosphates  are  derived  largely  from  the  food,  only 
a  small  proportion  resulting  from  metabolism.  The 
normal  daily  output  of  phosphoric  acid  is  about  2.5 
to  3.5  gm. 

The  urinary  phosphates  are  of  two  kinds:  alkaline, 
which  make  up  two-thirds  of  the  whole,  and  include  the 
phosphates  of  sodium  and  potassium;  and  earthy,  which 
constitute  one-third,  and  include  the  phosphates  of  cal- 
cium and  magnesium.  Earthy  phosphates  are  fre- 
quently thrown  out  of  solution  in  neutral  and  alkaHne 
urines,  and  as  "amorphous  phosphates"  form  a  very 
common  sediment.  This  sediment  seldom  indicates  an 
excessive  excretion  of  phosphoric  acid.  It  is  usually 
merely  an  evidence  of  diminished  acidity  of  the  urine,  or 
of  an  increase  in  the  proportion  of  phosphoric  acid  elimi- 
nated as  earthy  phosphates.  This  form  of  "phospha- 
turia"  is  most  frequent  in  neurasthenia  and  hysteria. 
When  the  urine  undergoes  ammoniacal  decomposition, 
some  of  the  ammonia  set  free  combines  with  magnesium 
phosphate  to  form  ammoniomagnesium  phosphate 
("triple  phosphate"),  which  is  deposited  in  typical 
crystalHne  form  (see  p.  185). 

Excretion  of  phosphates  is  increased  by  a  diet  rich  in 
nucleins;  in  active  metaboHsm;  in  certain  nervous  and 
mental  disorders;  in  leukemia;  and  in  phosphatic  dia- 
betes, an  obscure  disturbance  of  metabolism  (not  related 
to  diabetes  mellitus)  which  is  associated  with  an  increase 


no 


THE   URINE 


in  the  output  of  phosphates  up  to  lo  gm.  or  more  in 
twenty-four  hours.  Phosphates  are  decreased  in  chronic 
diseases  with  lowered  metabolism;  in  hepatic  cirrhosis 
and  acute  yellow  atrophy;  in  pregnancy,  owing  to  de- 
veloping fetal  bones;  and  in  nephritis,  owing  to  kidney 
impermeability. 

Quantitative  estimation  does  not  furnish  much  of 
definite  cUnical  value.  The  centrifugal  method  is  the 
most  convenient. 

TABLE   FOR  THE  ESTIMATION  OF  PHOSPHATES  AFTER 
CENTRIFUGATION 

Showing  bulk-percentages  of  uranyl  phosphate  (^H\UO^POi)  and  the  cor- 
responding grainmetric  percentages  and  grains  per  ounce  of  phosphoric 
acid  {P,0^).—{Purdy.) 


Bulk-per- 
centage of 
H(U0„)P04. 

Percentage 
P.O5. 

Gr.  Per  Oz. 
P.O5. 

i 

Bulk-per- 
centage of 
H(U0.,)P04. 

Percentage 
P2O5. 

Gr.  Per  0«. 
P2O5. 

i 

0.02 

O.I 

II 

0.14 

0.67 

I 

0.04 

0.19 

12 

0.15 

0.72 

li 

P-04S 

0.22 

13 

0.16 

0.77 

2 

0.05 

0.24 

14 

0.17 

0.83 

2j 

o-oSS 

0.26 

IS 

0.18 

0.86 

3, 

0.06 

0.29 

16 

0.19 

0.91 

3i 

0.065 

0.31 

17 

0.2 

0.96 

4. 

0.07 

0.34 

18 

0.21 

I. 

4J 

0.07s 

0.36 

19 

0.22 

1.06 

5 

0.08 

0.38 

20 

0.23 

I.I 

6 

0.09 

0.43 

21 

0.24 

IIS 

7 

0.1 

0.48 

1         22 

0.25 

1.2 

8 

0.1 1 

053 

23 

0.26 

1.25 

9 

0.12 

0.58 

24 

0.27 

1-3 

lO 

0.13 

0.62 

25 

0.28 

1-35 

Bulk-percentage  to  be  read  from  graduation  on  the  side  of  the  tube. 


Purdy's  Centrifugal  Method. — ^Take  10  c.c.  urine  in  the 
graduated  tube,  add  2  c.c.  of  50  per  cent,  acetic  acid,  and 
3  c.c.  of  5  per  cent,  uranium  nitrate  solution.  Mix;  let 
stand   a  few  mmutes,   and    revolve  for   three    minutes   at 


CHEMIC   EXAMINATION 


III 


I200  revolutions  a  minute.  Each  o.i  c.c.  of  precipitate  is 
I  per  cent,  by  bulk.  The  percentage  corresponding  of 
phosphoric  acid  by  weight  is  found  by  consulting  the  table 
on  page  no. 

3.  Sulphates.^ — The  urinary  sulphates  are  derived 
partly  from  the  food,  especially  meats,  and  partly  from 
body  metabolism.  The  normal  output  of  sulphuric  acid 
is  about  1.5  to  3  gm.  daily.  It  is  increased  in  condi- 
tions associated  with  active  metabolism,  and  in  general 
may  be  taken  as  a  rough  index  of  protein  metabolism . 

Quantitative  estimation  of  the  total  sulphates  yields 
little  of  clinical  value. 

TABLE  FOR  THE  ESTIMATION  OF  SULPHATES  AFTER 
CENTRIFUGATION 
Showing  the  hulk-percentages  of  barium  sulphate  (BaSO^)  and  the  cor- 
responding gravimetric  percentages  and  grains  per  fluidounce  of  sul- 
phuric acid  (5O3). — (Purdy.) 


Bulk-per- 
centage of 
BaSO*. 

Percentage 
SO3. 

Gr.  Per  Oz. 
SO3. 

1     Bulk-per- 
centage of 
BaSOi. 

Percentage 
SO3. 

Gr.  Per  Oz. 
SO3. 

i 

0.04 

0.19 

2} 

0-55 

2.64 

} 

0.07 

0-34 

2j 

0.61 

2-93 

1 

O.I 

0.48 

2| 

0.67 

3.22 

^ 

0.13 

0.62    • 

3 

0-73 

3-S 

1 

0.16 

0.77 

3i 

0.79 

3-79 

i 

0.19 

0.91 

^t 

0.85 

4.08 

i 

0.22 

1.06 

3l 

0.91 

4-37 

I 

0.25 

I.I 

4 

0.97 

4.66 

r\ 

0.31 

1.49 

4i 

1.03 

4.94 

^ 

0-37 

1.78 

4J 

1.09 

5-23 

i| 

0-43 

2.06 

4l 

i-iS 

5-52 

2 

0.49 

2-35 

5 

1. 21 

S.81   . 

Bulk-percentage  to  be  read  from  graduation  on  the  side  of  the  tube. 


Purdy's  Centrifugal  Method. — Take  10  c.c.  urine  in  the 
graduated   tube   and  add   barium   chlorid   solution   to   the 


112  THE   URINE 

15  c.c.  mark.  This  consists  of  barium  chlorid,  4  parts; 
strong  hydrochloric  acid,  i  part;  and  distilled  water,  16 
parts.  Mix;  let  stand  a  few  minutes,  and  revolve  for  three 
minutes  at  1200  revolutions  a  minute.  Each  o.i  c.c.  of 
precipitate  is  i  per  cent,  by  bulk  The  percentage  by 
weight  of  sulphuric  acid  is  calculated  from  the  table  on 
page  III. 

About  nine-tenths  of  the  sulphuric  acid  is  in  com- 
bination with  various  mineral  substances,  chiefly  sodium, 
potassium,  calcium,  and  magnesium  {mineral  or  pre- 
formed sulphates).  One- tenth  is  in  combination  with 
certain  aromatic  substances,  which  are  mostly  products 
of  protein  putrefaction  in  the  intestine,  but  are  de- 
rived in  part  from  destructive  metabolism  {conjugate 
or  ethereal  sulphates).  Among  these  aromatic  substances 
are  indol,  phenol,  and  skatol.  By  far  the  most  impor- 
tant of  the  conjugate  sulphates  and  representative  of  the 
group  is  potassium  indoxyl  sulphate. 

Potassium  indoxyl  sulphate,  or  indican,  is  derived 
from  indol.  Indol  is  absorbed  and  oxidized  into  in- 
doxyl, which  combines  with  sulphuric  acid  and  potas- 
sium and  is  thus  excreted.  Under  normal  conditions 
the  amount  in  the  urine  is  small.  It  is  increased  by  a 
meat  diet. 

Unlike  the  other  ethereal  sulphates,  which  are  de- 
rived in  part  from  metabolism,  iridican  originates  prac- 
tically wholly  from  putrefactive  processes.  It  alone, 
therefore,  and  not  the  total  ethereal  sulphates,  can  be 
taken  as  an  index  of  such  putrefaction.  A  pathologic 
increase  is  called  indicanuria.     It  is  noted  in: 

(a)  Diseases  of  the  Small  Intestine. — This  is  by  far 
the  most  common  source.    Intestinal  obstruction  gives 


CHEMIC  EXAMINATION  II3 

the  largest  amounts  of  indican.  It  is  also  much  in- 
creased in  intestinal  indigestion — so-called  "bilious- 
ness"; in  inflammations,  especially  in  cholera  and  ty- 
phoid fever;  and  in  paralysis  of  peristalsis,  such  as 
occurs  in  peritonitis.  Simple  constipation  and  diseases 
of  the  large  intestine  alone  do  not  so  frequently  cause 
indicanuria. 

(b)  Diseases  of  the  stomach  associated  with  deficient 
hydrochloric  acid,  as  chronic  gastritis  and  gastric  cancer. 
Diminished  hydrochloric  acid  favors  intestinal  putre- 
faction. 

(c)  Diminished  Flow  of  Bile. — Since  the  bile  serves 
both  as  a  stimulant  to  peristalsis  and  an  intestinal  anti- 
septic, a  diminished  flow  from  any  cause  favors  occur- 
rence of  indicanuria. 

(d)  Decomposition  of  exudates  anywhere  in  the  body, 
as  in  empyema,  bronchiectasis,  and  large  tuberculous 
cavities. 

Detection  of  indican  depends  upon  its  decomposition 
and  oxidation  of  the  indoxyl  set  free  into  indigo-blue. 
This  change  sometimes  takes  place  spontaneously  in 
decomposing  urine,  causing  a  dirty  blue  color.  Crystals 
of  indigo  (see  Fig.  37)  may  be  found  both  in  the  sedi- 
ment and  the  scum. 

Obermayer's  Method. — ^Take  a  test-tube  about  one-third 
full  of  the  urine  and  add  an  equal  volume  of  Obermayer's 
reagent  and  a  few  cubic  centimeters  of  chloroform.  Mix  by 
inverting  a  few  times;  avoid  shaking  violently.  If  indican  be 
present  in  excess,  the  chloroform,  which  sinks  to  the  bottom, 
will  assvime  an  indigo-blue  color.  It  will  take  up  the  indigo 
more  quickly  if  the  urine  be  warm.  The  depth  of  color  indi- 
cates the  comparative  amount  of  indican  if  the  same  propor- 
8 


114  THE    URINE 

tions  of  urine  and  reagents  are  always  used,  but  one  should 
bear  in  mind  the  total  amount  of  urine  voided.  The  indican 
in  normal  urine  may  give  a  faint  blue  by  this  method.  Urine 
of  patients  taking  iodids  gives  a  reddish-violet  color,  which 
disappears  upon  addition  of  a  few  drops  of  strong  sodium 
hyposulphite  solution  and  shaking.  Occasionally  indigo- 
red  will  form  instead  of  indigo-blue.  This  resembles  the 
color  due  to  iodids,  but  does  not  disappear  when  treated 
with  sodium  hyposulphite.  Bile-pigments,  which  interfere 
with  the  test,  must  be  removed  if  present  (see  p.  87).    • 

Obermayer's  reagent  consists  of  strong  hydrochloric  acid 
(sp.gr.,  1. 19),  1000  parts,  and  ferric  chlorid,  2  parts.  This 
makes  a  yellow,  fuming  liquid  which  keeps  well. 

4.  Urea. — From  the  standpoint  of  physiology  urea  is 
the  most  important  constituent  of  the  urine.  It  is  the 
principal  waste-product  of  metabolism,  and  constitutes 
about  one-half  of  all  the  solids  excreted — about  20  to 
35  .gm.  in  twenty-four  hours.  It  represents  85  to  90 
per  cent,  of  the  total  nitrogen  of  the  urine,  and  its  quan- 
titative estimation  is  a  simple,  though  not  very  accurate, 
method  of  ascertaining  the  state  of  nitrogenous  excretion. 

This  is  true,  however,  only  in  normal  individuals 
upon  average  mixed  diet.  Under  pathologic  conditions, 
the  proportion  of  nitrogen  distributed  among  the  various 
nitrogen-containing  substances  undergoes  great  varia- 
tion. The  only  accurate  index  of  protein  metabolism 
is,  therefore,  the  total  output  of  nitrogen,  which  can  be 
estimated  by  the  Kjeldahl  method.  The  whole  subject 
of  "nitrogen  partition"  and  "nitrogen  equilibrium"  (re- 
lation of  excretion  to  intake)  is  an  important  one,  but 
is  out  of  the  province  of  this  book,  since  as  yet  it  con- 
cerns the  physiologic  chemist  more  than  the  clinician. 


CHEMIC   EXAMINATION  II 5 

It  may  be  helpful  to  state  here,  however,  that  upon  a  mixed 
diet  the  nitrogen  of  the  urine  is  distributed  about  as  follows: 
urea  nitrogen,  86.9  per  cent.;  ammonia  nitrogen,  4.4  per 
cent.;  creatinin  nitrogen,  3.6  per  cent.;  uric  acid  nitrogen, 
0.75  per  cent.;  "undetermined  nitrogen,"  chiefly  in  amino- 
acids,  4.3  per  cent. 

Normally,  the  amount  is  greatly  influenced  by  ex- 
ercise and  diet.  It  is  increased  by  copious  drinking 
of  water  and  administration  of  ammonium  salts  of 
organic  acids. 

Pathologically,  urea  is  increased  in  fevers,  in  diabetes 
when  acidosis  is  not  marked,  and  especially  during 
resolution  of  pneumonia  and  absorption  of  large  exu- 
dates. As  above  indicated,  when  other  factors  are  equal, 
the  amount  of  urea  indicates  the  activity  of  metabol- 
ism. In  deciding  whether  in  a  given  case  an  increase  of 
urea  is  due  to  increased  metabolism  the  relation  between 
the  amounts  of  urea  and  of  the  chlorids  is  a  helpful  con- 
sideration. The  amount  of  urea  is  normally  about  twice 
that  of  the  chlorids.  If  the  proportion  is  much  increased 
above  this,  increased  tissue  destruction  may  be  inferred, 
since  other  conditions  which  increase  urea  also  increase 
chlorids. 

In  general,  a  pathologic  decrease  in  amount  of  urea 
is  due  either  to  lessened  formation  within  the  body  or 
to  diminished  excretion.  Decreased  formation  of  urea 
occurs  in  diseases  of  the  liver  with  destruction  of  liver 
substance,  such  as  marked  cirrhosis,  carcinoma,  and  acute 
yellow  atrophy.  The  state  of  acidosis  Hkewise  decreases 
formation  of  urea,  because  nitrogen  which  would  other- 
wise be  built  into  urea  is  eliminated  in  the  form  of  am- 
monia (see  p.  125).    Retention  of  urea  occurs  in  most  cases 


ii6 


THE  URINE 


of  nephritis.  In  acute  nephritis  the  amount  of  urea  in 
the  urine  is  markedly  decreased,  and  a  return  to  normal 
denotes  improvement.  In  the  early  stages  of  chronic 
nephritis,  when  diagnosis  is  difficult,  it  is  usually  normal. 
In  the  late  stages,  when  diagnosis  is  comparatively  easy, 
it  is  decreased.  Hence  estimation  of  urea  is  of  little  help 
in  the  diagnosis  of  this  disease,  especially  when,  as  is  so 
frequently  the  case,  a  small  quantity  of  urine  taken  at 
random  is  used.    When,  however,  the  diagnosis  is  estab- 


Fig.  23. — Crystab  of  nitrate  of  urea  (upper  half)  and 
oxalate  of  urea  (lower  half)  (after  Funke). 


Fig.  24. — Doremus-Hinds'  are- 
ometer without  foot. 


lished,  estimations  made  at  frequent  intervals  under  the 
same  conditions  of  diet  and  exercise  are  of  much  value, 
provided  a  sample  of  the  mixed  twenty-four-hottr  urine 
he  used.  A  steady  decline  is  a  very  bad  prognostic  sign, 
and  a  sudden  marked  diminution  is  usually  a  forerunner 
of  uremia. 

The  presence  of  urea  can  be  shown  by  allowing  a  few 
drops  of  the  fluid  partially  to  evaporate  upon  a  slide,  and 
adding  a  small  drop  of  pure,  colorless  nitric  acid  or 


CHEMIC  EXAMINATION  II 7 

saturated  solution  of  oxalic  acid.  Crystals  of  urea 
nitrate  or  oxalate  (Fig.  23)  will  soon  appear  and  can  be 
recognized  with  the  microscope. 

Quantitative  Estimation. — The  hypobromite  method, 
which  is  generally  used  in  clinical  work,  is  very  simple, 
but  is  notoriously  inaccurate.  The  new  urease  methods 
are  much  more  accurate. 

(i)  Hypobromite  Method. — ^This  depends  upon  the  fact 
that  urea  is  decomposed  by  sodium  hypobromite  with  liber- 
ation of  nitrogen.  The  amount  of  urea  is  calculated  from 
the  volume  of  nitrogen  set  free.  Of  the  many  forms  of  appa- 
ratus devised  for  this  purpose,  that  of  Doremus-Hinds  (Fig. 
24)  is  probably  the  most  convenient. 

Pour  some  of  the  urine  into  the  smaller  tube  of  the  appa- 
ratus, then  open  the  stop-cock  and  quickly  close  it  so  as  to  fill 
its  lumen  with  urine.  Rinse  out  the  larger  tube  with  water 
and  fill  it  and  the  bulb  with  25  per  cent,  caustic  soda  solu- 
tion. Add  to  this  i  c.c.  of  bromin  by  means  of  a  medicine- 
dropper  and  mix  well.  This  prepares  a  fresh  solution  of 
sodium  hypobromite  with  excess  of  caustic  soda,  which  serves 
to  absorb  the  carbon  dioxid  set  free  in  the  decomposition 
of  urea.  When  handling  bromin,  keep  an  open  vessel  of 
ammonia  near  to  neutralize  the  irritant  fumes. 

Pour  the  urine  into  the  smaller  tube,  and  then  turn  the 
stop-cock  so  as  to  let  as  much  urine  as  desired  (usually  i  c.c.) 
run  slowly  into  the  hypobromite  solution.  When  bubbles 
have  ceased  to  rise,  read  off  the  height  of  the  fluid  in  the  large 
tube  by  the  graduations  upon  its  side.  This  gives  the  amount 
by  weight  of  urea  in  the  urine  added,  from  which  the  amount 
excreted  in  twenty-four  hours  can  easily  be  calculated.  If 
the  urine  contains  much  more  than  the  normal  amount,  it 
should  be  diluted. 

This  method  has  fallen  into  disrepute  largely  because  of 


Il8  THE   URINE 

inconstant  results,  and  because  it  gives  more  nearly  the  total 
nitrogen  than  the  urea.  According  to  Robinson  and  Miiller 
the  discrepancies  are  due  to  insufficient  mixing  of  urine  and 
hypobromite  and  can  be  obviated  by  gentle  shaking  after 
the  first  vigorous  reaction  is  over.  Results  are  then  con- 
stant, but  too  high,  owing  to  decomposition  of  other  nitrog- 
enous constituents;  and  they  find  that  with  normal  urine 
the  ureometer  reading  multiplied  by  0.917  gives  the  true 
amount  of  urea. 

To  avoid  handling  pure  bromin,  which  is  disagreeable, 
Rice's  solutions  may  be  employed: 

(a)  Bromin 31  gm.; 

Potassium  bromid 31    " 

Distilled  water 250  c.c.j 

(b)  Sodium  hydroxid 100  gm.; 

Distilled  water 250  c.c. 

Equal  parts  of  these  solutions  are  mbced  and  used  for  the 
test.  The  bromin  solution  must  be  kept  in  a  tightly  stop- 
pered bottle  or  it  will  rapidly  lose  strength. 

(2)  Urease  Method. — In  brief,  this  consists  in  the  conver- 
sion of  urea  into  ammonium  carbonate  by  urease,  a  ferment 
first  extracted  by  Takeuchi  from  the  soy  bean  in  1909.  The 
amount  of  urea  is  calculated  from  the  amount  of  ammonia 
which  is  liberated  from  the  carbonate,  and  transferred  by  an 
air  current  to  a  standard  acid  solution,  and  this  titrated  with 
a  standard  alkali  solution.  The  method  was  first  employed 
by  Marshall.  Van  Slyke  and  Cullen  have  recently  intro- 
duced certain  improvements  which  render  it  more  suitable 
for  clinical  laboratory'  use.  Although  the  method  is  some- 
what complicated,  it  requires  no  apparatus  e.xcept  that  shown 
in  Fig.  25',  a  suction  filter-pump  which  can  be  attached  to 
the  water  faucet,  and  a  buret. 

'  The  apparatus  may  be  obtained  complete  from  E.  Greiner  &  Co., 
45  Cliff  Street,  New  York  City. 


CHEMIC   EXAMINATION 


119 


Method. — Since  the  ammonia  from  the  pre-existing  am- 
monium salts  of  the  urine  as  well  as  that  derived  from  urea  is 
carried  over  into  the  acid,  it  is  necessary  to  carry  through  two 
tests,  in  one  of  which  urease  is  used  and  in  the  other  omitted. 


To  suction  pump 


Fig.  25. — Apparatus  for  urease  method  of  estimating  urea. 


The  difference  between  the  results  obtained  in  the  two  rep- 
resents the  ammonia  from  urea  alone. 

(i)  For  urea  plus  ammonia: 
•  (a)  Dilute  5  c.c.  of  urine  to  50  c.c. 

(b)  In  tube  A    (Fig.  25)  place  5  c.c.  diluted  urine,  i  c.c. 
enzyme  solution,  and  i  dro])  caprylic  alcohol.     (The  last  is 


I20  THE   URmE 

added  to  prevent  foaming;  0.5-1  c.c.  toluol,  amyl  alcohol,  or 
kerosene  may  be  substituted.) 

(c)  Close  tube  A  and  let  stand  for  fifteen  minutes  for 
enzyme  to  act. 

(d)  In  tube  B  place  5  c.c.  ^  hydrochloric  or  sulphuric  acid, 
20  c.c.  distilled  water,  i  drop  of  i  per  cent,  aqueous  solution 
of  alizarin  sodium  sulphonate  to  serve  as  indicator,  and  i  drop 
cap ry lie  alcohol. 

(e)  Connect  tube  A  with  B  and  B  with  the  filter-pump, 
which  is  attached  to  the  water  faucet. 

(/)  Pass  the  air  current  (by  opening  water  faucet)  for  a 
half-minute.  This  will  carry  over  into  the  acid  any  ammonia 
that  may  have  escaped  from  the  mrine  into  the  air  space  in  A. 

(g)  Open  tube  A  and  pour  into  it  4  or  5  grams  of  dry 
potassium  carbonate. 

(h)  Close  A  and  pass  the  air  current  imtil  all  the  anmionia 
has  been  carried  over  into  the  acid.  This  will  require  from 
five  to  fifty  minutes,  depending  upon  the  rate  of  the  current. 
The  correct  time  can  be  determined  for  each  pump  by  trial. 

(i)  Titrate  the  acid  in  B  with  ^  sodium  hydroxid  from  a 
buret.  A  red  color  indicates  the  end-point.  The  difference 
between  the  amount  of  ^  acid  originally  taken  (5  c.c.)  and  the 
amount  of  ^  alkali  solution  used  in  the  titration  is  the  amount 
of  acid  which  has  been  neutralized  by  the  ammonia.  Since 
each  cubic  centimeter  of  the  -^  acid  solution  represents  0.0017 
gram  ammonia,  the  number  of  cubic  centimeters  of  the  ^  acid 
neutralized  by  the  ammonia  is  multiplied  by  the  factor  0.0017 
to  give  the  number  of  grams  of  ammonia  derived  from  the  urea 
plus  the  ammonium  salts  in  the  o.j  c.c.  of  urine  used;  and  this 
product  by  200  to  give  the  number  of  grams  from  100  c.c.  urine. 

When  extreme  accuracy  is  essential,  the  following  varia- 
tions should  be  introduced:  (i),  the  ammonia  of  the  air 
should  be  removed  by  passing  the  air  through  a  tube  of  weak 
sulphuric  acid  before  it  enters  tube  A;  (2),  a  second  tube  of 
standard  acid  should  be  interposed  between  B  and  the  sue- 


CHEMIC   EXAMINATION  121 

tion-pump  to  catch  any  ammonia  which  may  escape  neutral- 
ization in  B;  and  (3),  fiftieth-normal  alkali  solution  should  be 
employed  instead  of  decinormal,  thus  ensuring  greater  accu- 
racy in  titration. 

Preparation  of  Enzyme  Solution. — Dissolve  2  grams  urease 
powder/  0.6  gram  dipotassium  hydrogen  phosphate,  and  0.4 
gram  potassium  dihydrogen  phosphate  in  10  c.c.  water.  It 
forms  an  opalescent  solution,  which  will  keep  about  two 
weeks  if  covered  with  a  layer  of  toluol. 

(2)  For  ammonia  alone: 

This  requires  a  second  pair  of  tubes  which  is  connected  in 
series  with  the  first,  i.  e.,  interposed  between  the  first  pair 
and  the  suction-pump  in  such  manner  that  the  air  passes 
from  tube  B  of  the  first  pair  to  tube  A  of  the  second. 

The  technic  is  exactly  the  same  as  that  described  in  the 
preceding  paragraphs,  except  that  5  c.c.  of  undiluted  urine 
are  used  and  the  enzyme  solution  is  omitted  in  step  (b).  The 
number  of  cubic  centimeters  of  the  ^  acid  neutralized  by  the 
ammonia  is  multiplied  by  the  factor  0.0017  to  give  the  num- 
ber of  grams  of  ammonia  in  the  5  c.c.  of  urine  used;  and  this 
product  by  20  to  give  the  number  of  grams  of  ammonia  in 
100  c.c.  urine. 

This  is  essentially  Folin's  method  for  ammonia. 

(3)  Calculations: 

Find  by  the  two  methods  just  given: 

(i)  The  amount  of  ammonia  derived  from  the  urea 

plus  the  ammonium  salts  of  100  c.c.  urine. 
(2)  The  amount  of  ammonia  derived  from  the  ammo- 
nium salts,  only,  in  100  c.c.  urine. 
The  difference  between  (i)  and  (2)  gives  the  ammonia  de- 
rived from  the  urea  alone  in  100  c.c.  urine.     Each  gram  of 

.  ^  This  can  be  obtained  from  the  Arlington  Chemical  Co. ,  Yonkers, 
N.  Y.  It  keeps  indefinitely.  It  can  also  be  purchased  in  i-gram 
portions  mixed  with  the  correct  amount  of  phosphate  ready  to  be  dis- 
solved in  the  appropriate  quantity  of  water. 


122  THE    URINE 

ammonia  represents  1.7647  gram  of  urea.  If,  for  example, 
the  difference  between  (i)  and  (2)  is  1.5  grams,  then 
1.5  X  1.7647  =  2.647  grams  of  urea  in  100  c.c.  urine.  From 
this  the  amount  in  the  twenty-four-hour  sample  is  calcu- 
lated. 

The  above  method  is  particularly  useful  in  estimating 
urea  in  blood  and  cerebrospinal  fluid.  Van  Slyke  and  CuUen 
use  3  c.c.  of  fresh  blood  or  fluid,  measured  with  an  accurate 
pipet  into  a  loo-c.c.  test-tube  containing  i  c.c.  of  3  per 
cent,  potassium  citrate  to  prevent  clotting.  To  this  are 
added  0.5  c.c.  urease  solution  and  2  or  3  drops  caprylic  alco- 
hol, and  the  method  carried  out  as  above  described. 

5.  Uric  acid  is  the  most  important  of  a  group  of 
substances,  called  purin  bodies,  which  are  derived  chiefly 
from  the  nucleins  of  the  food,  exogenous  uric  acid,  and 
from  metabolic  destruction  of  the  nuclei  of  the  body, 
endogenous  uric  acid.  The  daily  output  of  uric  acid  is 
about  0.4  to  I  gm.  The  amount  of  the  other  purin 
bodies  together  is  about  one-tenth  that  of  uric  acid. 
Excretion  of  these  substances  is  greatly  increased  by  a 
diet  rich  in  nucleins,  as  sweetbreads  and  liver. 

Uric  acid  exists  in  the  urine  in  the  form  of  urates, 
chiefly  of  sodium  and  potassium,  which  in  concentrated 
urines  are  readily  thrown  out  of  solution  and  constitute 
the  familiar  sediment  of  "amorphous  urates."  This, 
together  with  the  fact  that  uric  acid  is  frequently  de- 
posited as  crystals,  constitutes  its  chief  interest  to  the 
practitioner.  It  is  a  very  common  error  to  consider  these 
deposits  as  evidence  of  excessive  excretion. 

Pathologically,  the  greatest  increase  of  uric  acid  occurs 
in  leukemia,  where  there  is  extensive  destruction  of 
leukocytes,  and  in  diseases  with  active  destruction  of  the 


CHEMIC  EXAMINATION  1 23 

liver  and  other  organs  rich  in  nuclei.  There  is  generally 
an  increase  during  x-ray  treatment.  Uric  acid  is  de- 
creased before  an  attack  of  gout  and  increased  for  several 
days  after  it,  but  its  etiologic  relation  is  still  uncertain. 
An  increase  is  also  noted  in  acute  fevers. 

Quantitative  Estimation  of  Purin  Bodies. — There  is  no 
accurate  method  which  is  simple  enough  for  chnical 
purposes.  Of  clinical  methods,  the  two  given  here  are 
most  satisfactory.  They  are  based  upon  the  same  prin- 
ciple: precipitation  and  removal  of  phosphates,  and  then 
precipitation  of  purin  bodies  with  silver  nitrate  which  is 
strongly  ammoniated  in  order  to  hold  silver  chlorid  in 
solution.  The  amount  of  purin  bodies  is  calculated  from 
the  bulk  of  the  silver-purin,  which  in  Cook's  method  is 
thrown  down  by  the  centrifuge,  and  in  Hall's  is  allowed 
to  settle  for  twenty-four  hours.  The  urine  must  be 
albumin  free. 

(i)  Cook's  Method. — In  a  centrifuge  tube  take  10  c.c.  urine 
and  add  about  i  gm.  (about  i  c.c.)  sodium  carbonate  and  i 
or  2  c.c.  strong  ammonia.  Shake  until  the  soda  is  dissolved. 
The  earthy  phosphates  will  be  precipitated.  Centrifugalize 
thoroughly  and  pour  off  all  the  clear  fluid  into  a  graduated 
centrifuge  tube.  Add  2  c.c.  ammonia  and  2  c.c.  ammoniated 
silver  nitrate  solution.  Let  stand  a  few  minutes,  and  re- 
volve in  the  centrifuge  until  the  bulk  of  precipitate  remains 
constant.  Each  tV  c.c.  of  sediment  represents  o.ooii76gm. 
purin  bodies. 

Ammoniated  silver  nitrate  solution  is  prepared  by  dissolving 
5  gm.  of  silver  nitrate  in  100  c.c.  distilled  water,  and  adding 
ammonia  until  the  solution  clouds  and  again  becomes  clear. 

(2)  Hall's  Method. — The  instrument  is  shown  in  Fig.  26. 
Close  the  stop-cock,  introduce  90  c.c.  urine  and  20  c.c.  of 


124 


THE  URINE 


u 

tern 

mx 

M 


E90 


;7o 


E60 


E50 


E« 


the  magnesia  solution,  and  mix  by  inverting  a  few  times. 

Open  the  stop-cock  and  let  the  instrument  stand  for  about 

ten  minutes,  or  until  the  precipitated  phosphates  have  set- 
tled into  the  lower  chamber.  Then  close 
the  stop-cock,  and  pour  in  ammonia  ted 
silver  nitrate  solution  until  the  level  of 
the  fluid  reaches  the  loo  c.c.  mark. 
Mix  well,  and  if  any  white  precipitate 
of  silver  chlorid  persists,  bring  it  into 
solution  by  adding  a  few  drops  of  am- 
monia. Stand  the  instrument  in  the 
dark  for  twenty-four  hours  and  read  off 
the  bulk  of  the  precipitate.  The  cor- 
responding percentage  of  purin  nitro- 
gen is  found  by  reference  to  a  table 
which  accompanies  the  instrument. 
Albumin  must  be  removed  before 
making  the  test. 

The  magnesia  mixture  is  prepared  by 
dissoving  lo  gm.  of  magnesium  chlorid 
in  75  c.c.  of  water  and  adding  lo  gm.  of 
ammonium  chlorid  and  loo  c.c.  strong 
ammonium  hydroxid.  If  a  precipitate 
forms,  it  is  dissolved  by  further  addition 
of  ammonia.  Add  water  to  bring  the 
volume  to  200  c.c.  and  finally  add  10 
gm.  of  finely  powdered  talcum. 
The  ammoniated  silver  nitrate  solution  used  in  Hall's  method 

consists  of  silver  nitrate,  i  gram;  ammonium  hydroxid,  100 

c.c;  talcum,  5  grams;  distilled  water,  100  c.c. 


Fig.  26. — Hall's  purinom- 
eter. 


Quantitative  Estimation  of  Uric  Acid. — Ruhemann's 
method,  while  not  accurate,  will  probably  answer  for 
clinical  work. 


CHEMIC  EXAMINATION 


125 


Ruhemann's  Method  for  Uric  Acid. — ^The 
urine  must  be  slightly  acid.  By  means  of 
a  pipet  fill  Ruhemann's  tube  (Fig.  27)  to 
the  mark  S  with  the  indicator,  carbon  disul- 
phid,  so  that  the  lowest  part  of  the  menis- 
cus is  on  a  level  with  the  mark,  as  indi- 
cated in  Fig.  27.  Next  add  Ruhemann's 
reagent  until  the  base  of  the  upper  arch  of 
the  meniscus  is  level  with  the  mark  /. 
The  carbon  disulphid  will  assume  a  violet 
color.  Add  the  urine,  a  small  quantity  at  a 
time,  closing  the  tube  with  the  glass  stopper 
and  shaking  vigorously  after  each  addition, 
imtil  the  disulphid  loses  every  trace  of  its 
violet  color  and  becomes  pure  white.  This 
completes  the  test.  Toward  the  end  the 
reagent  should  be  added  a  very  little  at  a 
time,  and  the  shaking  should  be  prolonged 
in  order  not  to  pass  the  end-point.  The 
figure  in  the  right-hand  column  of  figures 
corresponding  to  the  top  of  the  fluid  gives 
the  amount  of  uric  acid  in  parts  per  thou- 
sand. The  presence  of  diacetic  acid  inter- 
feres with  the  test,  as  do  also,  to  some  ex- 
tent, bile  and  albumin.  Diacetic  acid  can 
be  driven  off  by  boiling;  bile-pigment  and 
albimiin  are  removed  as  described  else- 
where (see  pp.  87  and  142). 

Ruhemann's  reagent  consists  of  iodin,  0.5 
gm.;  potassiimi  iodid,  1.25  gm.;  absolute 
alcohol,  7.5  gm.;  glycerin,  5  gm.;  distilled 
water  to  100  c.c. 

6.  Ammonia. — A  small  amount  of 
ammonia,  combined  with  hydrochloric, 
phosphoric,   and   sulphuric   acids,  is  al- 


126  THE   URINE 

ways  present.  Estimated  as  NH;,,  the  normal  average 
is  about  0.7  gm.  in  twenty-four  hours.  This  represents 
4  to  5  per  cent,  of  the  total  nitrogen  of  the  urine, 
ammonia  standing  next  to  urea  in  this  respect. 

Under  ordinary  conditions,  most  of  the  ammonia 
which  results  from  the  metabolic  processes  is  trans- 
formed into  urea.  When,  however,  acids  are  present 
in  excess,  either  from  ingestion  of  mineral  acids  or 
from  abnormal  production  of  acids  within  the  body 
(as  in  fevers,  diabetes,  pernicious  vomiting  of  preg- 
nancy, delayed  chloroform-poisoning,  etc.),  ammonia 
combines  with  them  and  is  so  excreted,  urea  being  cor- 
respondingly decreased.  It  is  thus  that  the  body  pro- 
tects itself  against  acid  intoxication.  A  marked  in- 
crease of  ammonia  is,  therefore,  important  chiefly  as 
an  index  of  the  tendency  to  acidosis,  particularly  that 
associated  with  the  presence  of  diacetic  and  oxybutyric 
acids. 

In  diabetes  melUtus  ammonia  elimination  may  reach 
4  or  5  gm.  daily.  It  is  likewise  markedly  increased  in 
pernicious  vomiting  of  pregnancy,  but  not  in  neroous 
vomiting;  and  in  conditions  in  which  the  power  to  syn- 
thesize urea  is  interfered  with,  notably  cirrhosis  and 
other  destructive  diseases  of  the  liver  and  conditions 
associated  with  deficient  oxygenation. 

Quantitative  Estimation. — The  urine  must  be  fresh, 
since  decomposition  increases  the  amount  of  ammonia. 
The  following  method  is  satisfactory  for  cUnical  pur- 
poses, though  subject  to  some  inaccuracies.  It  includes 
amino-acids  with  the  ammonia,  hence  gives  figures  that 
are  a  little  too  high.  Folin's  method  for  ammonia,  which 
is  more  accurate,  was  given  on  page  121. 


CHEMIC  EXAMINATION  1 27 

Ronchese-Malfatti  Formalin  Test. — This  depends  upon 
the  fact  that  when  formahn  is  added  to  the  urine  the  am- 
monia combines  with  it,  forming  hexamethylenamin.  The 
acids  with  which  the  ammonia  was  combined  are  set  free, 
and  their  quantity,  ascertained  by  titration  with  sodium 
hydroxid,  indicates  the  amount  of  ammonia. 

Take  10  c.c.  of  the  urine  in  a  beaker  or  evaporating  dish, 
add  50  c.c.  water  and  10  drops  of  0.5  per  cent,  alcoholic  solu- 
tion of  phenolphthalein.  Neutralize  by  adding  a  weak 
caustic  soda  or  sodium  carbonate  solution  until  a  permanent 
pink  color  appears.  To  5  c.c.  formalin  add  15  c.c.  water 
and  neutralize  in  the  same  way.  Pour  the  formalin  into  the 
urine.  The  pink  color  at  once  disappears,  owing  to  libera- 
tion of  acids.  Now  add  decinormal  sodiimi  hydroxid  solution 
from  a  buret  until  the  pink  color  just  returns.  Each  cubic 
centimeter  of  the  decinormal  solution  used  in  this  titration 
corresponds  to  0.0017  g"^-  of  NH3.  This  must  be  multi- 
plied by  ID  to  obtain  the  percentage  from  which  the  twenty- 
four-hour  elimination  of  ammonia  is  calculated. 

The  method  is  more  complicated,  but  distinctly  more 
accurate,  when  carried  out  as  suggested  by  E.  W.  Brown: 
Treat  60  c.c.  of  urine  with  3  gm.  of  basic  lead  acetate,  stir 
well,  let  stand  a  few  minutes,  and  filter.  This  removes  cer- 
tain interfering  nitrogenous  substances.  Treat  the  filtrate 
with  2  gm.  neutral  potassium  oxalate,  stir  well,  and  filter. 
Take  10  c.c.  of  the  filtrate,  add  50  c.c.  water  and  15  gm. 
neutral  potassium  oxalate,  and  proceed  with  the  ammonia 
estimation  as  above  outlined. 

B.  Abnormal  Constituents 

Those  substances  which  appear  in  the  urine  only  in 

pathologic  conditions  are  of  much  more  interest  to  the 

clinician  than  are  those  which  have  just  been  discussed. 

Among  them  are:  proteins,  sugars,  the  acetone  bodies. 


128  THE   URINE 

bile,  hemoglobin,  and  the  diazo  substances.  The  "pan- 
creatic reaction"  and  detection  of  drugs  in  the  urine  will 
also  be  discussed  under  this  head. 

1.  Proteins. — Of  the  proteins  which  may  appear 
in  the  urine,  serum-albumin  and  serum-globulin  are  the 
most  important.  Mucin,  proteose,  and  a  few  others  are 
found  occasionally,  but  are  of  less  interest. 

(i)  Serum-albumin  and  Serum-globulin. — These  two 
proteins  constitute  the  so-called  "urinary  albumin." 
They  usually  occur  together,  have  practically  the  same 
significance,  and  both  respond  to  all  the  ordinary  tests 
for  "albumin." 

Their  presence,  or  albuminuria,  is  probably  the  most 
important  pathologic  condition  of  the  urine.  It  is 
either  accidental  or  renal.  The  physician  can  make  no 
greater  mistake  than  to  regard  all  cases  of  albuminuria 
as  indicating  kidney  disease. 

Accidental  or  false  albuminuria  is  due  to  admixture 
with  the  urine  of  albuminous  fluids,  such  as  pus,  blood, 
and  vaginal  discharge.  The  microscope  will  usually 
reveal  its  nature.  It  occurs  most  frequently  in  pyelitis, 
cystitis,  and  chronic  vaginitis. 

Renal  albuminuria  refers  to  albumin  which  has  passed 
from  the  blood  into  the  urine  through  the  walls  of  the 
kidney  tubules  or  the  glomeruli. 

Albuminuria  sufficient  to  be  recognized  by  clinical 
methods  probably  never  occurs  as  a  physiologic  condi- 
tion, the  so-called  physiologic  albuminuria  appearing 
only  under  conditions  which  must  be  regarded  as 
abnormal.  Among  these  may  be  mentioned  excessive 
muscular  exertion  in  those  unaccustomed  to  it;  exces- 
sive ingestion  of  proteins;  prolonged  cold  baths,  and 


CHEinC   EXAMINATION  1 29 

childbirth.  In  these  conditions  the  albuminuria  is 
slight  and  transient. 

There  are  certain  other  forms  of  albuminuria  which 
have  still  less  claim  to  be  called  physiologic,  but  which 
are  not  always  regarded  as  pathologic.  Among  these 
are  cyclic  albuminuria,  which  regularly  recurs  at  a  certain 
period  of  the  day,  and  orthostatic  or  postural  albuminuria, 
which  appears  only  when  the  patient  is  standing.  They 
are  rare  and  of  obscure  origin,  and  occur  for  the  most 
part  in  neurasthenic  subjects  during  adolescence.  It  is 
noteworthy  in  this  connection  that  nephritis  sometimes 
begins  with  a  cyclic  albuminuria. 

In  pathologic  conditions  and  in  most,  at  least,  of  the 
"functional"  conditions  just  enumerated,  renal  al- 
buminuria may  be  referred  to  one  or  more  of  the  foUow- 
lowing  causes.  In  nearly  all  cases  it  is  accompanied  by 
tube-casts. 

(a)  Changes  in  the  blood  which  render  its  albumin 
more  diffusible,  as  in  severe  anemias,  purpura,  and 
scurvy.    Here  the  albumin  is  small  in  amount. 

(b)  Changes  in  circulation  in  the  kidney,  either  anemia 
or  congestion,  as  in  excessive  exercise,  chronic  heart 
disease,  and  pressure  upon  the  renal  veins.  The  quan- 
tity of  albumin  is  usually,  but  not  always,  small.  Its 
presence  is  constant  or  temporary,  according  to  the 
cause.  Most  of  the  causes,  if  continued,  will  produce 
organic  changes  in  the  kidney. 

(c)  Organic  Changes  in  the  Kidney. — These  include 
the  inflammatory  and  degenerative  changes  commonly 
grouped  together  under  the  name  of  nephritis,  and  also 
renal  tuberculosis,  neoplasms,  and  cloudy  sweUing  due 
to  irritation  of  toxins  and  drugs.     The  amount  of  al- 

9 


130  THE   URINE 

buniin  eliminated  in  these  conditions  varies  from  minute 
traces  to  20  gm.,  or  even  more,  in  the  twenty-four  hours, 
and,  except  in  acute  processes,  bears  little  relation  to 
the  severity  of  the  disease.  In  acute  and  chronic  paren- 
chymatous nephritis  the  quantity  is  usually  very  large. 
In  chronic  interstitial  nephritis  it  is  small — frequently 
no  more  than  a  trace.  It  is  small  in  cloudy  swelling 
from  toxins  and  drugs,  and  variable  in  renal  tuber- 
culosis and  neoplasms.  In  amyloid  disease  of  the 
kidney  the  quantity  is  usually  small,  and  serum- 
globulin  may  be  present  in  especially  large  propor- 
tion, or  even  alone.  Roughly  distinctive  of  serum- 
globulin  is  the  appearance  of  an  opalescent  cloud  when 
a  few  drops  of  the  urine  are  dropped  into  a  glass  of  dis- 
tilled water. 

Detection  of  albumin  depends  upon  its  precipitation 
by  chemicals  or  coagulation  by  heat.  There  are  many 
tests,  but  none  is  entirely  satisfactory,  because  other 
substances  as  well  as  albumin  are  precipitated.  The 
most  common  source  of  error  is  mucin.  When  any 
considerable  amount  of  mucin  is  present  it  can  be 
removed  by  acidifying  with  acetic  acid  and  filtering. 
The  tests  given  here  are  widely  used  and  can  be 
recommended.  They  make  no  distinction  between 
serum-albumin  and  serum-globulin.  Usually  the  best 
time  to  detect  albumin  is  in  the  evening  or  a  few 
hours  after  a  meal. 

//  is  very  important  that  urine  to  be  tested  for  albumin 
be  rendered  clear  by  filtration  or  centrifugation.  This 
is  too  often  neglected  in  routine  work.  When  ordinary 
methods  do  not  suffice,  it  can  usually  be  cleared  by 
shaking  up  with  a  little  purified  talc  or  animal  charcoal 


CHEMIC   EXAMINATION  131 

and  filtering.     If  the  urine  is  alkaline,  sufficient  acetic 
acid  should  be  added  to  make  it  acid  to  litmus. 

(i)  Trichloracetic  Acid  Test. — The  reagent  consists  of  a 
saturated  aqueous  solution  of  trichloracetic  acid  to  which 
magnesium  sulphate  is  added  to  saturation.  A  simple 
saturated  solution  of  the  acid  may  be  used,  but  addition  of 
magnesium  sulphate  favors  precipitation  of  globulin,  and, 
by  raising  the  specific  gravity,  makes  the  test  easier  to  apply. 

Take  a  few  cubic  centimeters  of  the  reagent  in  a  test-tube 
or  conical  test  glass,  hold  the  tube  or  glass  in  an  inclined 
position,  and  run  the  urine  gently  in  by  means  of  a  pipet  or 
medicine-dropper,  so  that  it  will  form  a  layer  on  top  of  the 
reagent  without  mixing  with  it.  If  albumin  be  present,  a 
white,  cloudy  ring  will  appear  where  the  two  fluids  come  in 
contact.  The  ring  can  be  seen  most  clearly  if  viewed  against 
a  black  background,  and  one  side  of  the  tube  or  conical  glass 
may  be  painted  black  for  this  purpose. 

This  is  an  extremely  sensitive  test,  but,  unfortunately, 
both  mucin  and  proteoses  respond  to  it;  urates,  when  abun- 
dant, may  give  a  confusing  white  ring,  and  the  reagent  is 
comparatively  exp>ensive.  It  is  not  much  used  in  routine 
work  except  as  a  control  to  the  less  sensitive  tests. 

A  convenient  and  satisfactory  instrument  for  applying 
this  or  any  of  the  contact  tests  is  sold  under  the  name  of 
"horismascope"  (Fig.  28).  In  Boston's  method  of  perform- 
ing the  ring  tests  the  fluids  are  brought  into  contact  in  a 
glass  pipet,  which  is  immersed  first  in  the  lighter  fluid  and 
then  (after  wiping  the  outside  of  the  pipet)  in  the  heavier. 
This  is  widely  used  and  is  convenient  for  routine  testing 
of  a  large  number  of  urines,  but  it  cannot  be  recommended 
for  accuracy,  owing  to  the  small  diameter  of  the  column 
of-  fluid. 

(2)  Robert's  Test. — ^The  reagent  consists  of  pure  nitric 
acid,  I  part,  and  saturated  aqueous  solution  of  magnesium 


132  THE  URINE 

sulphate,  5  parts.    It  is  applied  in  the  same  way  as  the  pre- 
ceding test. 

Albumin  gives  a  white  ring,  which  varies  in  density  with 
the  amount  present.  A  similar  white  ring  may  be  produced 
by  primary  proteose,  thymol,  and  resinous  drugs.  White  rings 
or  cloudiness  in  the  urine  above  the  zone  of  contact  may  result 
from  excess  of  urates  or  mucus.    Colored  rings  near  the  junc- 


Fig.  28. — Horismascope:  adding  the  reagent. 

tion  of  the  fluids  may  be  produced  by  iodids,  urinary  pig- 
ments, bile,  or  indican. 

Robert's  test  is  one  of  the  best  for  routine  work,  although 
the  various  rings  are  apt  to  be  confusing  to  the  inexperienced. 
It  is  more  sensitive  than  Heller's  test,  of  which  it  is  a  modi- 
fication, and  has  the  additional  advantage  that  the  reagent 
is  not  so  corrosive. 


CHEMIC  EXAMINATION  I33 

(3)  Ulrich's  test  avoids  the  somewhat  confusing  colored 
rings.  The  reagent  consists  of  saturated  solution  of  common 
salt,  98  c.c. ;  glacial  acetic  acid,  2  c.c.  It  must  be  perfectly 
clear.  Boil  a  few  cubic  centimeters  of  this  fluid  in  a  test- 
tube,  and  immediately  overlay  with  the  urine  as  in  the  pre- 
ceding tests.  Albumin  and  globulin  give  a  white  ring  at  the 
zone  of  contact. 

(4)  Purdy's  Heat  Test. — ^Take  a  test-tube  two-thirds  full 
of  urine,  add  about  one-sixth  its  volume  of  saturated  solution 
of  sodiimi  chlorid,  and  5  to  10  drops  of  50  per  cent,  acetic  acid. 
Mix,  and  boil  the  upper  inch.  A  white  cloud  in  the  heated 
portion  shows  the  presence  of  albumin. 

This  is  a  valuable  test  for  routine  work.  It  is  simple, 
sufficiently  accurate  for  clinical  purposes,  and  has  practically 
no  fallacies.  Addition  of  the  salt  solution,  by  raising  the 
specific  gravity,  prevents  precipitation  of  miccin.  Proteose 
may  produce  a  white  cloud,  which  disappears  upon  boiling 
and  reappears  upon  cooling. 

(5)  Heat  and  Nitric  Acid  Test. — ^This  is  one  of  the  oldest 
of  the  albumin  tests,  and,  if  properly  carried  out,  one  of  the 
best.  Boil  about  5  c.c.  of  filtered  urine  in  a  test-tube  and 
add  I  to  3  drops  of  concentrated  nitric  acid.  A  white 
cloud  or  flocculent  precipitate  (which  usually  appears  during 
the  boiling,  but  if  the  quantity  be  very  small  only  after  addi- 
tion of  the  acid)  denotes  the  presence  of  albumin.  A  similar 
white  precipitate,  which  disappears  upon  addition  of  the 
acid,  is  due  to  earthy  phosphates.  The  acid  should  not  be 
added  before  boiling,  and  the  proper  amount  should  always 
be  used;  otherwise,  part  of  the  albumin  may  fail  to  be  pre- 
cipitated or  may  be  redissolved. 

Quantitative  Estimation. — The  gravimetric,  which  is 
the  most  reliable  method,  is  too  elaborate  for  clinical 
work.    Both  Esbach's,  which  is  very  widely  used,  and 


134 


THE   URINE 


Purdy's  centrifugal  method  give  fair  results,  but  Tsu- 

chiya's  modification  of  the  Esbach  method  is  preferable 

to  either. 

(i)  Esbach's  Method. — The  urine  must 
be  clear,  of  acid  reaction,  and  not  con- 
centrated. Always  filter  before  testing, 
and,  if  necessary',  add  acetic  acid  and 
dilute  with  water.  Esbach's  tube  (Fig.  29) 
is  essentially  a  test-tube  with  a  mark  U 
near  the  middle,  a  mark  R  near  the  top, 
and  graduations  §,  i,  2,  3,  etc.,  near  the 
bottom.  Fill  the  tube  to  the  mark  U  with 
urine  and  to  the  mark  R  with  the  reagent. 
Close  with  a  rubber  stopper,  invert  slowly 
several  times,  and  set  aside  in  a  cool  place. 
At  the  end  of  twenty-four  hours  read  off 
the  height  of  the  precipitate.  This  gives 
the  amount  of  albumin  in  grams  per  liter, 
and  must  he  divided  by  10  to  obtain  the  per- 
centage. 

Esbach's   reagent  consists  of  picric  acid, 
I    gm.,    citric   acid,    2  gm.,   and  distilled 
water,  to  make  100  c.c. 
(2)  Tsuchiya's  Method. — This  is  carried  out  in  the  same 

manner  as  the  Esbach  method,  using  the  following  reagent: 

Phosphotungstic  acid 1.5  gm.; 

Alcohol  (q6  per  cent.) 95.0  c.c; 

Concentrated  hydrochloric  acid 5.0   " 


-Esbach's 


albuminometer, 
proved  form. 


The  urine  should  be  diluted  to  a  specific  gravity  not  exceed- 
ing 1 .008.  The  method  is  said  to  be  much  more  accurate  than 
the  original  Esbach  method,  particularly  with  small  quanti- 
ties of  albumin. 

(3)  Purdy's    Centrifugal    Method. — This    is    detailed    in 
the  table  on  opposite  page.    Since  10  c.c.  of  urine  were  used, 


CHEMIC   EXAMINATION 


135 


PURDY'S    QUANTITATIVE    METHOD    FOR   ALBUMIN    IN 
URINE    (CENTRIFUGAL). 

Table  showing  the  relation  between  the  volumetric  and  gravimetric  percentage 

of  albumin  obtained  by  means  of  the  centrifuge  with  radius  of  six 

and  three-quarter  inches  ;  rate  of  speed,  1500  revolutions 

per  minute  ;    time,  three  minutes. 


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0.448 

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0.51 

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0.146 

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0.521 

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31 

Test. — Three  cubic  centimeters  of  10  per  cent,  solution  of  ferrocyanid  of 
potassium  and  2  cubic  centimeters  of  50  per  cent,  acetic  acid  are  added  to  10  cubic 
centimeters  of  the  urine  in  the  percentage  tube  and  stood  aside  for  ten  minutes, 
then  placed  in  the  centrifuge  and  revolved  at  rate  of  speed  and  time  as  stated  at 
hgad  of  the  table.  If  albumin  is  excessive,  dilute  the  urine  with  water  until 
volume  of  albumin  falls  below  10  per  cent.  Multiply  result  by  the  number 
of  dilutions  employed  before  using  the  table. 


136  THE  URINE 

each  0.1  c.c.  of  precipitate  is  i  per  cent,  by  bulk.  Instead 
of  the  ferrocvanid  and  acetic  acid  Tsuchiya's  solution  may 
be  used. 

(2)  Mucin. — Traces  of  the  substances  (mucin,  mu- 
coid, etc.)  which  are  loosely  classed  under  this  name  are 
present  in  normal  urine;  increased  amounts  are  observed 
in  irritations  and  inflammations  of  the  mucous  mem- 
brane of  the  urinary  tract.  They  are  of  interest  chiefly 
because  they  may  be  mistaken  for  albumin  in  most  of 
the  tests.  If  the  urine  be  diluted  with  water  and  acidi- 
fied with  acetic  acid,  the  appearance  of  a  white  cloud  in- 
dicates the  presence  oi  mucin. 

True  mucin  is  a  gly co-protein,  and  upon  boiling  with 
an  acid  or  alkali,  as  in  Fehling's  test,  yields  a  carbohy- 
drate substance  which  reduces  copper. 

(3)  Proteoses. — These  are  intermediate  products  in 
the  digestion  of  proteins  and  are  frequently,  although 
incorrectly,  called  albumoses.  Two  groups  are  generally 
recognized:  primary  proteoses,  which  are  precipitated 
upon  half-saturation  of  their  solutions  with  ammonium 
sulphate;  and  secondary  proteoses,  which  are  precipitated 
only  upon  complete  saturation. 

The  secondary  proteoses  have  been  observed  in  the 
urine  in  febrile  and  malignant  diseases  and  chronic  sup- 
purations, during  resolution  of  pneumonia,  and  in  many 
other  conditions,  but  their  clinical  significance  is  in- 
definite. In  pregnancy,  albumosuria  may  be  due  to 
absorption  of  amniotic  fluid. 

Primary  proteoses  are  rarely  encountered  in  the  urine. 
The  protein  known  as  the  ''Bence-Jones  body"  was 
originally  classed  under  this  head,  but  its  true  nature  is 


CHEMIC  EXAMINATION  137 

uncertain.  It  is  regarded  as  practically  pathognomonic 
of  multiple  myeloma.  It  has  recently  been  found  in  a 
number  of  cases  of  chronic  leukemia,  of  both  lymphatic 
and  myelogenous  types. 

The  proteoses  are  not  coagulable  by  heat,  but  are  precipi- 
tated by  such  substances  as  trichloracetic  acid  and  phos- 
photungstic  acid.  The  primary  proteoses  alone  are  pre- 
cipitated by  nitric  acid. 

Proteoses  may  be  detected  by  acidifying  the  urine  with 
acetic  acid,  boiling  and  filtering  while  hot  to  remove  mucin, 
albumin,  and  globulin,  and  testing  the  filtrate  by  the  tri- 
chloracetic acid  test.  As  above  indicated,  the  nitric  acid 
test,  and  half  and  complete  saturation  with  ammonium  sul- 
phate, will  separate  the  two  groups. 

To  detect  Bence- Jones'  protein  the  urine  is  acidified  with 
acetic  acid  and  gently  heated  in  a  water-bath.  If  this  sub- 
stance be  present,  the  urine  will  begin  to  be  turbid  at  about 
40°  C.  and  a  precipitate  will  form  at  about  60°  C.  As  the 
boiling-point  is  reached  the  precipitate  wholly  or  partially 
dissolves.    It  reappears  upon  cooling. 

2.  Sugars. — Various  sugars  may  at  times  be  found  in 
the  urine.  Dextrose  is  by  far  the  most  common,  and  is 
the  only  one  of  clinical  importance.  Levulose,  lactose, 
and  some  others  are  occasionally  met  with. 

(i)  Dextrose  (Glucose). — It  is  probable  that  traces  of 
glucose,  too  small  to  respond  to  the  ordinary  tests,  are 
present  in  the  urine  in  health.  Its  presence  in  appre- 
ciable amount  constitutes  ''glycosuria." 

Transitory  glycosuria  is  unimportant,  and  may  occur 
in  many  conditions,  as  after  general  anesthesia  and 
administration  of  certain  drugs,  in  pregnancy,  and 
following  shock  and  head  injuries.     It  may  also  occur 


138  THE    URINE 

after  eating  excessive  amounts  of  carbohydrates  (ali- 
mentary glycosuria).  The  "assimilation  limit"  varies 
with  different  individuals  and  with  different  conditions  of 
exercise.  It  also  depends  upon  the  kind  of  carbohydrate. 
The  normal  for  glucose  is  about  icx3  to  150  gm.  When 
more  than  this  amount  is  taken  at  one  time  some  of  it 
will  be  excreted  in  the  urine.  Excretion  lasts  for  a  period 
of  four  or  five  hours. 

Persistent  glycosuria  has  been  noted  in  brain  injuries 
involving  the  floor  of  the  fourth  ventricle.  As  a  rule, 
however,  persistent  glycosuria  is  diagnostic  of  diabetes 
melhtus,  of  which  disease  it  is  the  essential  symptom. 
The  amount  of  glucose  eliminated  in  diabetes  is  usually 
considerable,  and  is  sometimes  very  large,  reaching  500 
gm.,  or  even  more,  in  twenty-four  hours,  but  it  does  not 
bear  any  uniform  relation  to  the  severity  of  the  disease. 
Glucose  may,  on  the  other  hand,  be  almost  or  entirely 
absent  temporarily. 

Detection  of  Dextrose. — If  albumin  be  present  in  more 
than  traces,  it  must  be  removed  by  boiling  and  filtering. 

(i)  Haines'  Test. — Take  about  4  c.c.  of  Haines'  solution 
in  a  test-tube,  boil,  examine  carefully  for  a  precipitate,  and, 
if  none  is  present,  add  6  or  8  drops  of  urine.  A  heav\^  yellow 
or  red  precipitate,  which  settles  readily  to  the  bottom,  shows 
the  presence  of  sugar.  Neither  precipitation  of  phosphates, 
as  a  light,  fiocculent  sediment,  nor  simple  decolorization 
of  the  reagent  should  be  mistaken  for  a  positive  reaction. 

This  is  one  of  the  best  of  the  copper  tests,  all  of  which 
depend  upon  the  fact  that  in  strongly  alkaline  solutions 
glucose  reduces  cupric  hydrate  to  cuprous  hydrate  (yellow)  or 
cuprous  oxid  (red).  They  are  somewhat  inaccurate,  because 
they  make  no  distinction  between  glucose  and  less  common 


CHEMIC   EX.4MINATI0N  139 

forms  of  sugar;  because  certain  normal  substances,  when 
present  in  excess,  especially  mucin,  uric  acid,  and  creatinin, 
may  reduce  copper,  and  because  many  drugs — e.  g.,  chloral, 
chloroform,  copaiba,  acetanilid,  benzoic  acid,  morphin,  sul- 
phonal,  salicylates — are  eliminated  as  copper-reducing  sub- 
stances. To  minimize  these  fallacies  dilute  the  urine,  if  it 
be  concentrated;  do  not  add  more  than  the  specified  amount 
of  urine,  and  do  not  boil  after  the  urine  is  added.  If  chloro- 
form has  been  used  as  a  preservative,  it  should  be  removed 
by  boiling  the  urine  before  making  the  test. 

Haines^  solution  is  prepared  as  follows:  Completely  dis- 
solve 2  gm.  pure  copp>er  sulphate  in  16  c.c.  distilled  water,  and 
add  16  c.c.  pure  glycerin;  mix  thoroughly,  and  add  156  c.c. 
liquor  potassae.     The  solution  keeps  well. 

(2)  Fehling's  Test. — ^Two  solutions  are  required — one 
containing  34.64  gm.  pure  crystalline  copper  sulphate  in 
500  c.c.  distilled  water;  the  other,  173  gm.  Rochelle  salt  and 
loo  gm.  potassium  hydroxid  in  500  c.c.  distilled  water.  MLx 
equal  parts  of  the  two  solutions  in  a  test-tube,  dilute  with 
3  or  4  volumes  of  water,  and  boil.  Add  the  urine  a  little  at  a 
time,  heating,  but  not  boiling,  between  additions.  In  the 
presence  of  glucose  a  heavy  red  or  yellow  precipitate  will 
appear.  The  quantity  of  urine  should  not  exceed  that  of  the 
reagent.  The  fallacies  mentioned  under  Haines'  Test  apply 
equally  to  this. 

(3)  Benedict's  Test. — This  new  test  promises  to  displace 
all  other  reduction  tests  for  glucose.  The  reagent  is  said  to 
be  ten  times  as  sensitive  as  Haines'  or  Fehling's,  and  not  to 
be  reduced  by  uric  acid,  creatinin,  chloroform,  or  the  alde- 
hyds.     It  consists  of: 

Copper  sulphate  (pure  crystallized) 17.3  gm.; 

Sodium  or  potassium  citrate i73-o  " 

Sodium  carbonate  (crystallized) 200.0  " 

(or  100  gm.  of  the  anhydrous  salt). 
Distilled  water,  to  make looo.o  c.c. 


I40  TECE  UKENE 

Dissolve  the  citrate  and  carbonate  in  700  c.c.  of  water, 
with  the  aid  of  heat,  and  filter.  Dissolve  the  copper  in  100  c.c. 
of  water  and  pour  slowly  into  the  first  solution,  stirring  con- 
stantly. Cool,  and  make  up  to  one  liter.  The  reagent  keeps 
indefinitely. 

Take  about  5  c.c.  of  this  reagent  in  a  test-tube,  and  add 
8  or  10  drops  {not  more)  of  the  urine.  Heat  to  vigorous 
boiling,  keep  at  this  temperature  for  one  or  two  minutes, 
and  allow  to  cool  slowly.  In  the  presence  of  glucose  the 
entire  body  of  the  solution  will  be  filled  with  a  precipitate, 
which  may  be  red,  yellow,  or  green  in  color.  When  traces 
only  of  glucose  are  present,  the  precipitate  may  appear  only 
upon  cooling.  In  the  absence  of  glucose,  the  solution  re- 
mains clear  or  shows  only  a  faint,  bluish  precipitate,  due  to 
urates. 

(4)  Phenylhydrazin  Test. —  Kowarsky's  Method. — ^The  fol- 
lowing directions  include  certain  modifications  which  have 
recently  been  worked  out  by  C.  S.  Bluemel  in  the  writer's 
laboratory:  In  a  \Ndde  test-tube  take  5  drops  pure  phenyl- 
hydrazin, 10  drops  glacial  acetic  acid,  and  i  c.c.  saturated 
solution  of  sodium  chlorid.  A  curdy  mass  results.  Add  3 
or  4  c.c.  of  the  urine  and  4  or  5  c.c.  of  water.  Boil  vigor- 
ously for  two  to  three  minutes.  The  annoying  bumping  can 
be  reduced  or  obviated  by  shaking  continually,  or,  much 
better,  by  placing  in  the  test-tube  a  number  of  pieces  of 
glass  tubing,  varying  in  length  from  i^  to  3  inches,  so  as 
to  produce  an  organ-pipe  effect.  The  volume  of  fluid  re- 
maining should  be  2  to  3  c.c.  Set  aside  to  cool,  or  if  the 
glass  tubes  were  used  pour  the  fluid  into  another  hot 
test-tube  and  allow  to  cool.  Examine  the  sediment  with 
the  microscope,  using  a  two-thirds  objective.  If  glucose 
be  present,  characteristic  crystals  of  phenylglucosazone  will 
be  seen.  These  are  yellow,  needle-like  crystals  arranged 
mostly  in  clusters  or  in  sheaves  (Fig.  30) .  When  traces  only 
of  glucose  are  present,  the  crystals  may  not  app>ear  for  one- 


CHEMIC  EXAMINATION 


141 


half  hour  or  more.  The  best  crystals  are  obtained  when  the 
fluid  is  cooled  very  slowly.  It  must  not  be  agitated  during 
cooling.  The  test-tubes  and  pieces  of  tubing  can  be  cleaned 
when  necessary  by  boiling  in  a  solution  of  caustic  soda  or 
acetic  acid. 

This  is  an  excellent  test  for  clinical  work.  Bluemel  finds 
that  when  applied  as  above  directed,  with  the  tubing  to  pre- 
vent bumping,  it  will  readily  detect  0.025  per  cent,  of  glucose 
in  urine,  the  crystals  appearing  in  three  to  four  hours.     The 


Fig.  30. — Crystals  of  phenylglucosazone  from  diabetic  urine — Kowarsky's  test  (Xsoo)- 


test  has  practically  no  fallacies  excepting  levulose,  which  is  a 
fallacy  for  all  the  ordinary  tests.  Other  carbohydrates  which 
are  capable  of  forming  crystals  with  phenylhydrazin  are  ex- 
tremely unlikely  to  do  so  when  the  test  is  applied  directly 
to  the  urine.  Even  if  not  used  routinely,  this  test  should 
always  be  resorted  to  when  the  copper  tests  give  a  positive 
reaction  in  doubtful  cases. 

Quantitative  Estimation. — In  quantitative  work  Feh- 
ling's  solution,  for  so  many  years  the  standard,  has  been 


142  THE   URINE 

largely  displaced  by  Purdy's,  which  avoids  the  heavy 
precipitate  that  so  greatly  obscures  the  end-reaction  in 
Fehling's  method.  The  older  method  is  still  preferred 
by  many,  and  both  are,  therefore,  given.  The  newer 
method  of  Benedict  is  likewise  included,  since  it  appears 
to  be  more  exact  and  more  satisfactory  than  any  other 
titration  method  available  for  sugar  work.  Should  the 
urine  contain  much  glucose,  it  must  be  diluted  before 
making  any  quantitative  test,  allowance  being  made  for 
the  dilution  in  the  subsequent  calculation.  Albumin,  if 
present,  must  be  removed  by  acidifying  a  considerable 
quantity  of  urine  with  acetic  acid,  boiUng,  and  filtering. 
The  precipitate  should  then  be  washed  with  water  and 
the  washings  added  to  the  urine  to  bring  it  to  its  original 
volume. 

(i)  Purdy's  Method. — Take  exactly  35  c.c.  of  Purdy's 
solution  in  a  flask  or  beaker,  add  twice  its  volume  of  distilled 
water,  heat  to  boiling,  and,  still  keeping  the  solution  hot,  add 
the  urine  very  slowly  from  a  buret  until  the  blue  color  entirely 
disappears.  Read  otf  the  amount  of  urine  added;  considering 
the  strength  of  Purdy's  solution,  it  is  readily  seen  that  this 
amount  of  urine  contains  0.02  gm.  of  glucose,  from  which  the 
amount  in  the  twenty-four-hour  urine,  or  the  percentage,  can 
easily  be  calculated.  Example:  Supjjose  that  2.5  c.c.  of 
urine  discharged  the  blue  color  of  35  c.c.  of  Purdy's  solution. 
This  amount  of  urine,  therefore,  contains  exactly  0.02  gm. 
glucose,  and  the  percentage  is  obtained  from  the  equation: 
2.5  :  100  :  :  0.02  :  .v,  and  .v  equals  0.8  per  cent.  If,  then,  the 
twenty-four-hour  quantity  of  urine  were  3000  c.c,  the 
twenty-four-hour  elimination  of  glucose  would  be  found  as 
follows:  100  :  3000  :  :  0.8  :  .v,  and  x  equals  24  gm. 

It  will  be  found  that  after  the  test  is  completed  the  blue 


CHEMIC   EXAMINATION  143 

color  slowly  returns.  This  is  due  to  reoxidation,  and  should 
not  be  mistaken  for  incomplete  reduction. 

A  somewhat  simpler  application  of  this  method,  which  is 
accurate  enough  for  most  clinical  purposes,  is  as  follows:  Take 
8f  c.c.  (roughly,  9  c.c.)  of  Purdy's  solution  in  a  large  test- 
tube,  dilute  with  an  equal  volume  of  water,  heat  to  boiling, 
and,  while  keeping  the  solution  hot  but  not  boiling,  add  the 
urine  drop  by  drop  from  a  medicine-dropper  until  the  blue 
color  is  entirely  gone.  Toward  the  end  add  the  drops  very 
slowly,  not  more  than  4  or  5  a  minute.  Divide  10  by  the 
number  of  drops  required  to  discharge  the  blue  color;  the 
quotient  will  be  the  percentage  of  glucose.  If  20  drops  were 
required,  the  percentage  would  be  10-^-20  =  0.5  per  cent. 
It  is  imperative  that  the  drops  be  of  such  size  that  20  of  them 
will  make  i  c.c.  Test  the  dropper  with  urine,  not  water.  If 
the  drops  are  too  large,  draw  out  the  tip  of  the  dropper;  if 
too  small,  file  oflf  the  tip. 

Purdy^s  solution  consists  of  pure  crystalline  copper  sulphate, 
4.752  gm.;  potassium  hydroxid,  23.5  gm.;  ammonia  (U.  S.  P.; 
sp.  gr.,  0.9),  350  c.c;  glycerin,  38  c.c;  distilled  water,  to  make 
1000  c.c.  Dissolve  the  copper  sulphate  and  glycerin  in  200 
c.c.  of  the  water  by  aid  of  gentle  heat.  In  another  200  cc 
of  water  dissolve  the  potassium  hydroxid.  Mix  the  two  solu- 
tions and,  when  cool,  add  the  ammonia.  Lastly,  bring  the 
whole  up  to  1000  c.c.  w'ith  distilled  water.  This  solution  is 
of  such  strength  that  the  copper  in  35  c.c.  will  be  reduced  by 
exactly  0.02  gm.  of  glucose. 

(2)  Fehling's  Method. — ^Take  10  c.c.  of  Fehling's  solution 
(made  by  mixing  5  c.c.  each  of  the  copper  and  alkaline  solu- 
tions described  on  page  139)  in  a  flask  or  beaker,  add  3 
or  4  volumes  of  water,  boil,  and  add  the  urine  very  slowly 
from  a  buret  until  the  solution  is  completely  decolorized, 
heating  but  not  boiling  after  each  addition. 

The  chief  objection  to  Fehling's  method  is  the  difficulty 
of  determining  the  end-point.    The  use  of  an  "outside  indi- 


144  THE   URINE 

cator,"  however,  obviates  this.  When  reduction  is  thought 
to  be  complete,  a  few  drops  of  the  solution  are  filtered  through 
a  fine-grained  filter-paper  on  to  a  porcelain  plate,  quickly 
acidified  with  acetic  acid,  and  mixed  with  a  drop  of  lo  per 
cent,  potassium  ferrocyanid.  Immediate  appearance  of  a 
red-brown  color  shows  the  presence  of  unreduced  copper. 

Fehling's  solution  is  of  such  strength  that  the  copper  in 
lo  c.c.  will  be  reduced  by  exactly  0.05  gm.  of  glucose.  There- 
fore, the  amount  of  urine  required  to  decolorize  the  test  solu- 
tion contains  just  0.05  gm.  glucose,  and  the  percentage  is 
easily  calculated. 

(3)  Benedict's  Method. — ^The  following  modification  of 
his  copper  solution  has  been  offered  by  Benedict  for  quanti- 
tative estimations. 

The  reagent  consists  of: 

Copper  sulphate  (pure  crystallized) 18.0  gm.; 

Sodium  carbonate  (crystallized) 200.0   " 

(or  100  gm.  of  the  anhydrous  salt). 

Sodium  or  potassium  citrate 200.0 

Potassium  sulphocyanate, 125.0 

Potassium  ferrocyanid  solution  (5  per  cent.) ...  5.0  c.c; 

Distilled  water,  to  make looo.o 

With  the  aid  of  heat  dissolve  the  carbonate,  citrate,  and 
sulphoc>'anate  in  about  800  c.c.  of  the  water  and  filter. 
Dissolve  the  copper  in  100  c.c.  of  water  and  pour  slowly 
into  the  other  fluid,  stirring  constantly.  Add  the  ferro- 
cyanid solution,  cool,  and  dilute  to  1000  c.c.  Only  the 
copper  need  be  accurately  weighed.  This  solution  is  of  such 
strength  that  25  c.c.  are  reduced  by  0.05  gram  glucose.  It 
keeps  well. 

To  make  a  sugar  estimation,  take  25  c.c.  of  the  reagent  in 
a  porcelain  evaporating  dish,  add  10  to  20  gm.  of  sodium 
carbonate  crystals  (or  one-half  this  weight  of  the  anhydrous 
salt)  and  a  small  quantity  of  powdered  pumice-stone  or  tal- 


CHEMIC   EXAMINATION 


145 


cum.  Heat  to  boiling,  and  add  the  urine  rather  rapidly 
from  a  buret  until  a  chalk-white  precipitate  forms  and  the 
blue  color  of  the  reagent  begins  to  fade.  After  this  point  is 
reached,  add  the  urine  a  few  drops  at  a  time  until  the  last 
trace  of  blue  just  disappears.  This  end-point  is  easily  recog- 
nized. During  the  whole  of  the  titration  the  mixture  must 
be  kept  vigorously  boiling.  Loss  by  evaporation  must  be 
made  up  by  adding  water.  The  quantity  of  urine  required 
to  discharge  the  blue  color  contains  exactly  0.05  gm.  glu- 
cose, and  the  percentage  contained  in  the  original  sample  is 
easily  calculated. 


Fig.  31. — Einhom's  saccharimeter. 

(4)  Fermentation  Method. — This  is  convenient  and  satis- 
factory, its  chief  disadvantage  being  the  time  required.  It 
depends  upon  the  fact  that  glucose  is  fermented  by  yeast  with 
evolution  of  CO2.  The  amount  of  gas  evolved  is  an  index  of 
the  amount  of  glucose.  No  preservative  must  have  been 
added.  Einhorn's  saccharimeter  (Fig.  31)  is  the  simplest 
apparatus. 

The  urine  must  be  so  diluted  as  to  contain  not  more  than 
I  per  cent,  of  glucose.  A  fragment  of  fresh  yeast-cake  about 
10 


146  THE  URINE 

the  size  of  a  split-pea  is  mixed  with  a  definite  quantity  of  the 
urine  measured  in  the  tube  which  accompanies  the  apparatus. 
It  should  form  an  emulsion  free  from  lumps  or  air-bubbles. 
The  long  arm  of  the  apparatus  is  then  filled  with  the  mLxture. 
At  the  end  of  fifteen  to  twenty-four  hours  fermentation  will 
be  complete,  and  the  percentage  of  glucose  can  be  read  ofif 
upon  the  side  of  the  tube.  The  result  must  then  be  multi- 
plied by  the  degree  of  dilution.  Since  yeast  itself  sometimes 
gives  off  gas,  a  control  test  must  be  carried  out  with  normal 
urine  and  the  amount  of  gas  evolved  must  be  subtracted 
from  that  of  the  test.  A  control  should  also  be  made  with  a 
known  glucose  solution  to  make  sure  that  the  yeast  is  active. 
It  has  recently  been  shown  that  yeast  can  split  off  carbon 
dioxid  from  amino-acids,  so  that  the  results  with  the  fermenta- 
tion method  are  likely  to  be  a  little  high. 

(5)  Robert's  Diflferential  Density  Method. — While  this 
method  gives  only  approximate  results,  it  is  convenient,  and 
requires  no  special  apparatus  but  an  accurate  urinometer. 
Mix  a  quarter  of  an  yeast-cake  with  about  100  c.c.  of  urine. 
Take  the  specific  gravity  and  record  it.  Set  the  urine  in  a 
warm  place  for  twenty-four  hours  or  until  fermentation  is 
complete.  Then  cool  to  the  temperature  at  which  the  specific 
gravity  was  originally  taken,  and  take  it  again.  The  differ- 
ence between  the  two  readings  gives  the  number  of  grains  of 
sugar  per  ounce,  and  this,  multiplied  by  0.234,  gives  the 
percentage  of  sugar.  If  the  original  reading  is  1.035,  ^^'^  that 
after  fermentation  is  1.020,  the  urine  contains  1.035  — 1.020 
=  15  grains  of  sugar  per  fluidounce ;  and  the  p)ercentage  equals 
15X0.234  =  3.5. 

(2)  Levulose,  or  fruit  sugar,  is  seldom  present  in 
urine  except  in  association  with  dextrose,  and  has  about 
the  same  significance.  According  to  von  Noorden,  its 
appearance  in  diabetes  indicates  an  advanced  case.    Its 


CHEMIC   EXAMINATION  147 

name  is  derived  from  the  fact  that  it  rotates  polarized 
light  to  the  left. 

The  normal  assimilation  limit  for  levulose  is  about  loo 
gm.  This  fact  is  used  in  the  Strauss  test  of  the  functional 
capacity  of  the  liver.  One  hundred  gm.  of  levulose  are 
given  upon  an  empty  stomach,  and  the  subsequent  ap- 
pearance of  levulose  in  the  urine  is  taken  as  evidence  of 
deficiency  of  the  glycogenic  function.  The  degree  of  the 
hepatic  derangement  is  measured  by  a  quantitative 
estimation. 

Detection  of  Levulose. — Levulose  responds  to  all  the 
tests  above  given  for  dextrose.  It  may  be  distinguished 
from  dextrose  by  the  following : 

Borchardt's  Test. — Mix  about  5  c.c.  each  of  the  urine  and 
25  per  cent,  hydrochloric  acid  (concentrated  HCl,  2  parts; 
water,  i  part)  in  a  test-tube  and  add  a  few  crystals  of  re- 
sorcinol.  Heat  to  boiling  and  boil  for  not  more  than  one-half 
minute.  In  the  presence  of  levulose  a  red  color  appears. 
Cool  in  running  water,  pour  into  a  beaker,  and  render  slightly 
alkaline  with  solid  sodium  or  potassium  hydroxid.  Return 
to  the  test-tube,  add  2  or  3  c.c.  of  acetic  ether,  and  shake. 
If  levulose  be  present,  the  ether  will  be  colored  yellow.  A 
similar  yellow  color  will  follow  administration  of  rhubarb 
and  senna. 

If  indican  be  present  the  test  must  be  modified  as  follows: 
Perform  Obermayer's  test  and  extract  the  indican  with  chloro- 
form. Reduce  the  acidity  of  the  indican-free  urine  by  adding 
one-third  its  volume  of  water,  add  a  few  crystals  of  resorcinol, 
and  proceed  with  Borchardt's  test. 

Quantitative  Estimation  of  Levulose. — The  methods 
are  the  same  as  for  dextrose  (see  p.  141).  It  reduces  cop- 
per to  the  same  extent. 


148  THE   URINE 

(3)  Lactose,  or  milk-sugar,  is  sometimes  present  in 
the  urine  of  nursing  women  and  in  that  of  women  who 
have  recently  miscarried.  It  is  of  interest  chiefly  be- 
cause it  may  be  mistaken  for  glucose.  It  reduces  copper, 
hut  does  not  ferment  with  yeast.  In  strong  solution  it  can 
form  crystals  with  phenyUiydrazin,  but  is  extremely 
unlikely  to  do  so  when  the  test  is  applied  directly  to  the 
urine. 

(4)  Maltose  and  cane-sugar  are  of  little  or  no  clinical 
importance.  Maltose  has  been  found  along  with  dex- 
trose in  diabetes.  It  reduced  copper,  0.074  gm.  being 
equivalent  to  25  c.c.  of  Benedict's  solution.  Cane-sugar 
(sucrose)  is  sometimes  added  to  the  urine  by  malingering 
patients.    It  does  not  reduce  copper. 

(5)  Pentoses. — These  sugars  are  so  named  because 
they  contain  5  atoms  of  oxygen.  Vegetable  gums 
form  their  chief  source.  They  reduce  copper  strongly 
but  slowly,  and  give  crystals  with  phenylhydrazin^  but 
do  not  ferment  with  yeast. 

Pentosuria  is  uncommon.  It  has  been  noted  after  in- 
gestion of  large  quantities  of  pentose-rich  substances, 
such  as  cherries,  plums,  and  fruit-juices,  and  is  said  to 
be  fairly  constant  in  habitual  use  of  morphin.  It  some- 
times accompanies  glycosuria  in  diabetes.  An  obscure 
chronic  form  of  pentosuria  without  clinical  symptoms 
has  been  observed.  The  pentose  excreted  in  these  cases 
is  believed  to  be  optically  inactive  arabinose,  although 
recent  work  indicates  that  ribose  is  present  in  some  cases 
at  least. 

Bial's  Orcinol  Test. — ^Dextrose  is  first  removed  by  fermen- 
tation.   About  5  c.c.  of  Bial's  reagent  are  heated  in  a  test- 


CHEMIC  EXAMINATION  I49 

tube,  and  after  removing  from  the  flame  the  urine  is  added 
drop  by  drop,  not  exceeding   20  drops  in  all.    The  appear- 
ance of  a  green  color  denotes  pentose. 
The  reagent  consists  of: 

Hydrochloric  acid  (30  per  cent.) 500  c.c; 

Ferric  chlorid  solution  (10  per  cent.) 25  drops; 

Orcinol i  gm. 

3.  Acetone  Bodies. — This  is  a  group  of  closely  related 
substances — acetone,  diacetic  acid,  and  beta-oxybutyric 
acid.  Acetone  is  derived  from  decomposition  of  diacetic 
acid,  and  this  in  turn  from  beta-oxybutyric  acid  by  oxida- 
tion. The  origin  of  beta-oxybutyric  acid  is  not  definitely 
known,  but  it  is  probable  that  its  chief,  if  not  its  only, 
source  is  in  some  obscure  metabolic  disturbance  with 
abnormal  destruction  of  fats.  The  three  substances 
generally  appear  in  the  urine  in  the  order  mentioned. 
When  the  disturbance  is  mild,  acetone  only  appears;  as  it 
becomes  more  marked,  diacetic  acid  is  added,  and  finally 
beta-oxybutyric  acid  appears.  The  presence  of  beta- 
oxybutyric  acid  in  the  blood  is  probably  the  chief  cause 
of  the  form  of  auto-intoxication  known  as  "acid  intoxi- 
cation." 

(i)  Acetone. — Minute  traces,  too  small  for  the  ordi- 
nary tests,  may  be  present  in  the  urine  under  normal 
conditions.  Larger  amounts  are  not  uncommon  in 
fevers,  gastro-intestinal  disturbances,  and  certain  ner- 
vous disorders.  A  notable  degree  of  acetonuria  has 
likewise  been  observed  in  pernicious  vomiting  of  preg- 
nancy and  in  eclampsia. 

-  Acetonuria  is  practically  always  observed  in  acid 
intoxication,  and,  together  with  diaceturia,  constitutes 
its  most  significant  diagnostic  sign.    A  similar  or  identi- 


ISO 


THE   XIRINE 


cal  toxic  condition,  always  accompanied  by  acetonuria 
and  often  fatal,  is  now  recognized  as  a  not  infrequent 
late  effect  of  anesthesia,  particularly  of  chloroform  anes- 
thesia. This  postanesthetic  toxemia  is  more  likely  to 
appear,  and  is  more  severe  when  the  urine  contains  any 
notable  amount  of  acetone  before  operation,  which  sug- 
gests the  importance  of  routine  examination  for  acetone 
in  surgical  cases. 


Fig.  32. — A  simple  distilling  apparatus. 


Acetone  is  present  in  considerable  amounts  in  many 
cases  of  diabetes  mellitus,  and  is  always  present  in  severe 
cases.  Its  amount  is  a  better  indication  of  the  severity 
of  the  disease  than  is  the  amount  of  sugar.  A  progres- 
sive increase  is  a  grave  prognostic  sign.  It  can  be  di- 
minished temporarily  by  more  liberal  allowance  of 
carbohydrates  in  the  diet. 

According  to  Folin,  acetone  is  present  in  only  small 


CHEMIC   EXAMINATION  151 

amounts  in  these  conditions,  the  substance  shown  by 
the  usual  tests,  particularly  after  distillation  of  the 
urine,  being  really  diacetic  acid.  In  this  connection, 
Frommer's  test  is  to  be  recommended,  since  it  does  not 
require  distillation,  and  does  not  react  to  diacetic  acid 
unless  too  great  heat  is  applied. 

Detection  of  Acetone. — The  urine  may  be  tested  di- 
rectly, but  it  is  much  better  to  distil  it  after  adding  a 
little  phosphoric  or  hydrochloric  acid  to  prevent  foaming, 
and  to  test  the  first  few  cubic  centimeters  of  distillate. 
A  simple  distilling  apparatus  is  shown  in  Fig.  32.  The 
test-tube  may  be  attached  to  the  delivery  tube  by  means 
of  a  two-hole  rubber  cork  as  shown,  the  second  hole 
serving  as  air  vent,  or,  what  is  much  less  satisfactory, 
it  may  be  tied  in  place  with  a  string.  Should  the  vapor 
not  condense  well,  the  test-tube  may  be  immersed  in  a 
glass  of  cold  water. 

When  diacetic  acid  is  present,  a  considerable  pro- 
portion will  be  converted  into  acetone  during  distilla- 
tion. 

(i)  Gunning's  Test. — To  a  few  cubic  centimeters  of  urine 
or  distillate  in  a  test-tube  add  a  few  drops  of  tincture  of  iodin 
and  of  ammonia  alternately  until  a  heavy  black  cloud  appears. 
This  cloud  will  gradually  clear  up  and,  if  acetone  be  present, 
iodoform,  usually  crystalline,  will  separate  out.  The  iodoform 
can  be  recognized  by  its  odor,  especially  upon  heating  (there 
is  danger  of  explosion  if  the  mixture  be  heated  before  the 
black  cloud  disappears),  or  by  detection  of  the  crystals  mi- 
croscopically. The  latter  only  is  dependable,  unless  one  has  an 
unusually  acute  sense  of  smell.  The  odor  of  iodin,  which  is 
also  present,  is  confusing.  Iodoform  crystals  are  yellowish 
sLx-pointed  stars  or  sLx-sided  plates  (Fi^.^^^)^  -^i^  1 

l-KYbnCi 


152  THE   URINE 

This  modification  of  Lieben's  test  is  less  sensitive  than  the 
original,  but  is  sufficient  for  all  clinical  work;  it  has  the  ad- 
vantage that  alcohol  does  not  cause  confusion,  and  especially 
that  the  sediment  of  iodoform  is  practically  always  crystalline. 
When  ay>plied  directly  to  the  urine,  phosphates  are  precipi- 
tated and  may  form  large  feathery,  star-shaped  crystals  which 
are  confusing  to  the  inexperienced.  Albiunin  prevents  for- 
mation of  the  crystals,  and  when  it  is  present,  the  urine  must 
be  distilled  for  the  test. 


P  ^«.i>(£?»^r^*- 


Fig-  33- — Iodoform  crystals  obtained  in  several  tests  for  acetone  by  Gunning's  method 
(X  about  600). 


(2)  Lange's  Test. — This  is  a  modification  of  the  well- 
known  Legal  test.  It  is  more  sensitive  and  gives  a  sharper 
end-reaction.  To  a  small  quantity  of  urine  add  about  one- 
twentieth  its  volume  (i  drop  for  each  i  c.c.)  of  glacial  acetic 
acid  and  a  few  drops  of  fresh  concentrated  aqueous  solution 
of  sodium  nitroprussid,  and  gently  run  a  little  ammonia  upon 
its  surface.  If  acetone  be  present,  a  purple  ring  will  form 
within  a  few  minutes  at  the  junction  of  the  two  fluids. 

(3)  Frommer's  Test. — This  test  has  proved  very  satis- 
factory in  the  hands  of  the  writer.  The  urine  need  not  be 
distilled.    Alkalinize  about  10  c.c.  of  the  urine  with  2  or  3  c.c. 


CHEMIC  EXAMINATION  1 53 

of  40  per  cent,  caustic  soda  solution,  add  10  or  12  drops  of 
10  per  cent,  alcoholic  solution  of  salicylous  acid  (salicyl 
aldehyd),  heat  the  upper  portion  to  about  70  C.  (it  should 
not  reach  the  boiling-point),  and  keep  at  this  temperature 
five  minutes  or  longer.  In  the  presence  of  acetone  an  orange 
color,  changing  to  deep  red,  appears  in  the  heated  portion. 

The  test  can  be  made  more  definite  by  adding  the  caustic 
soda  in  substance  (about  i  gm.),  and  before  it  goes  into 
solution  adding  the  salicyl  aldehyd  and  warming  the  lower 
portion. 

(2)  Diacetic  acid  occurs  in  the  same  conditions  as 
acetone,  but  has  more  serious  significance.  In  diabetes 
its  presence  is  a  grave  symptom  and  often  forewarns  of 
approaching  coma.  It  rarely  or  never  occurs  without 
acetone. 

Detection. — The  urine  must  be  fresh. 

(i)  Gerhardt's  Test. — To  a  few  cubic  centimeters  of  the 
urine  add  solution  of  ferric  chlorid  (about  10  per  cent.)  drop 
by  drop  until  the  phosphates  are  precipitated;  filter  and  add 
more  of  the  ferric  chlorid.  If  diacetic  acid  be  present,  the 
urine  will  assume  a  Bordeaux-red  color  which  disappears 
upon  boiling.  A  red  or  violet  color  which  does  not  disappear 
upon  boiling  may  be  produced  by  other  substances,  as  phenol, 
salicylates,  and  antipyrin.  Whenever  the  reaction  is  doubtful 
the  urine  should  be  distilled  and  the  distillate  tested  for  ace- 
tone. 

(2)  Lindemann's  Test. — ^To  about  10  c.c.  of  urine  add 
5  drops  30  per  cent,  acetic  acid,  5  drops  Lugol's  solution,  and 
2  or  3  c.c.  chloroform,  and  shake.  The  chloroform  does  not 
change  color  if  diacetic  acid  be  present,  but  becomes  reddish 
violet  in  its  absence.  This  test  is  claimed  by  its  advocates  to 
be  more  sensitive  and  more  reliable  than  Gerhardt's.    Uric 


154  THE    URINE 

acid  also  decolorizes  iodin,  and  if  much  is  present  double  the 
amount  of  Lugol's  solution  should  be  used. 

(3)  Oxybutyric  acid  has  much  the  same  significance 
as  diacetic  acid,  but  is  of  more  serious  import. 

Hart's  Test. — Remove  acetone  and  diacetic  acid  by  dilut- 
ing 20  c.c.  urine  with  20  c.c.  water,  adding  a  few  drops  of 
acetic  acid,  and  boiling  down  to  10  c.c.  To  this  add  10  c.c. 
water,  mix,  and  divide  between  two  test-tubes.  To  one  tube 
add  I  c.c.  of  hydrogen  peroxid,  warm  gently,  and  cool.  This 
transforms  ^J-oxybutyric  acid  to  acetone.  Now  apply  Lange's 
test  for  acetone  (see  p.  152)  to  each  tube.  A  positive  reaction 
in  the  tube  to  which  hydrogen  peroxid  has  been  added  shows 
the  presence  of  /3-oxybutyric  acid  in  the  original  sample  of 
urine. 

4.  Bile  appears  in  the  urine  in  all  diseases  which 
produce  jaundice,  often  some  days  before  the  skin  be- 
comes yellow;  and  in  many  disorders  of  the  liver  not 
severe  enough  to  cause  jaundice.  It  also  occurs  in  dis- 
eases with  extensive  and  rapid  destruction  of  red  blood- 
corpuscles.  Both  bile-pigment  and  bile  acids  may  be 
found.  They  generally  occur  together,  but  the  pigment 
is  not  infrequently  present  alone.  Bilirubin,  only,  oc- 
curs in  freshly  voided  urine,  the  other  pigments  (bili- 
verdin,  bilifuscin,  etc.)  being  produced  from  this  by 
oxidation  as  the  urine  stands.  The  acids  are  almost 
never  present  without  the  pigments,  and  are,  therefore, 
seldom  tested  for  clinically.  Crystals  of  bilirubin  (hem- 
atoidin)  (Fig.  37,  4)  may  be  deposited  in  heavily  bile- 
charged  urine. 

Detection  of  Bile-pigment. — Bile-pigment  gives  the 
urine  a  greenish-yellow,  yellow,  or  brown  color,  which 


CHEMIC   EXAMINATION  1 55 

upon  shaking  is  imparted  to  the  foam.  Cells,  casts,  and 
other  structures  in  the  sediment  may  be  stained  brown 
or  yellow.  This,  however,  should  not  be  accepted  as 
proving  the  presence  of  bile  without  further  tests. 

(i)  Smith's  Test. — Overlay  the  urine  with  tincture  of  iodin 
diluted  with  nine  times  its  volume  of  alcohol.  An  emerald- 
green  ring  at  the  zone  of  contact  shows  the  presence  of  bile- 
pigments.  It  is  convenient  to  use  a  conical  test-glass,  one 
side  of  which  is  painted  white. 

(2)  Gmelin's  Test. — This  consists  in  bringing  slightly 
yellow  nitric  acid  into  contact  with  the  vu-ine.  A  play  of 
colors,  of  which  green  and  violet  are  most  distinctive,  denotes 
the  presence  of  bile-pigment.  Colorless  nitric  acid  will  be- 
come yellow  upon  standing  in  the  sunlight.  The  test  may  be 
applied  in  various  ways:  by  overlaying  the  acid  with  the  urine; 
by  bringing  a  drop  of  each  together  upon  a  porcelain  plate; 
by  filtering  the  urine  through  thick  filter-paper,  and  touching 
the  paper  with  a  drop  of  the  acid;  and,  probably  best  of  all, 
by  precipitating  with  lime-water,  filtering,  and  touching  the 
precipitate  with  a  drop  of  the  acid.  In  the  last  method  bili- 
rubin is  carried  down  as  an  insoluble  calcium  compoimd 
which  concentrates  the  pigment  and  removes  interfering 
substances  such  as  indican. 

Detection  of  Bile  Acids. — Hay's  test  is  simple,  sensi- 
tive, and  fairly  reliable,  and  will,  therefore,  appeal  to 
the  practitioner.  It  depends  upon  the  fact  that  bile 
acids  lower  surface  tension.  Other  tests  require  isola- 
tion of  the  acids  for  any  degree  of  accuracy. 

"  Hay's  Test. — Upon  the  surface  of  the  urine,  which  must 
not  be  warm,  sprinkle  a  little  finely  powdered  sulphur.  If  it 
sinks  at  once,  bile  acids  are  present  to  the  amomit  of  o.oi 


156  THE   URINE 

per  cent,  or  more;  if  only  after  gentle  shaking,  0.0025  P^r 
cent,  or  more.  If  it  remains  floating,  even  after  gentle 
shaking,  bile  acids  are  absent.  It  is  said  that  urobilin  when 
present  in  large  amount  also  reduces  surface  tension. 

5.  Hemoglobin. — The  presence  in  the  urine  of  hemo- 
globin or  pigments  directly  derived  from  it,  accompanied 
by  few,  if  any,  red  corpuscles,  constitutes  hemoglobinuria. 
It  is  a  comparatively  rare  condition,  and  must  be  dis- 
tinguished from  hematuria,  or  blood  in  the  urine,  which 
is  common.  In  both  conditions  chemic  tests  will  show 
hemoglobin,  but  in  the  latter  the  microscope  will  reveal 
the  presence  of  red  corpuscles.  Urines  which  contain 
notable  amounts  of  hemoglobin  have  a  reddish  or  brown 
color,  and  may  deposit  a  sediment  of  brown,  granular 
pigment. 

Hemoglobinuria  occurs  when  there  is  such  extensive 
destruction  of  red  blood-cells  within  the  body  that  the 
liver  cannot  transform  all  the  hemoglobin  set  free  into 
bile-pigment.  The  most  important  examples  are  seen  in 
poisoning,  as  by  mushrooms  and  potassium  chlorate, 
in  scurvy  and  purpura,  in  malignant  malaria  (blackwater 
fever),  and  in  the  obscure  condition  known  as  "paroxys- 
mal hemoglobinuria."  This  last  is  characterized  by  the 
appearance  of  large  quantities  of  hemoglobin  at  inter- 
vals, usually  following  exposure  to  cold,  the  urine  re- 
maining free  from  hemoglobin  between  the  attacks. 

Detection. — Teichmann's  test  may  be  applied  to  the 
precipitate  after  boiling  and  filtering,  but  the  guaiac 
or  benzidin  test  will  be  found  more  convenient  in 
routine  work.  For  further  discussion  of  blood  tests  see 
page  306. 


CHEMIC  EXAMINATION  1 57 

Guaiac  Test. — Mix  a  few  cubic  centimeters  each  of  "ozon- 
ized" turpentine  and  a  fresh  i  :  60  alcoholic  solution  of  guaiac. 
The  guaiac  solution  may  be  freshly  prepared  by  dissolving 
a  pocket-knife-pointful  of  powdered  guaiac  in  4  or  5  c.c.  of 
alcohol.  Boil  a  few  cubic  centimeters  of  the  urine  in  a  test- 
tube,  cool,  and  gently  pour  the  guaiac- turpentine  mixture  on 
its  surface.  A  bright  blue  ring  will  appear  at  the  zone  of 
contact  within  a  few  minutes  if  hemoglobin  be  present. 
The  guaiac  should  be  kept  in  an  amber-colored  bottle.  Fresh 
turpentine  can  be  "ozonized"  by  allowing  it  to  stand  a  few 
days  in  an  open  vessel  in  the  sunlight.  Instead  of  turpentine, 
hydrogen  peroxid  may  be  used. 

This  test  is  very  sensitive,  and  a  negative  result  proves  the 
absence  of  hemoglobin.  Positive  results  are  not  conclusive, 
because  numerous  other  substances — few  of  them  likely  to  be 
foimd  in  the  urine — may  produce  the  blue  color.  That  most 
likely  to  cause  confusion  is  pus,  but  the  blue  color  produced 
by  it  disappears  upon  heating.  The  thin  film  of  copper 
often  left  in  a  test-tube  after  testing  for  sugar  may  give  the 
reaction,  as  may  also  the  fumes  from  an  open  bottle  of 
bromin. 

Benzidin  Test. — The  reagents  employed  are  hydrogen 
peroxid  and  a  saturated  solution  of  benzidin  in  glacial  acetic 
acid.  The  test  is  applied  in  exactly  the  same  manner  as  the 
guaiac  test  just  given.  It  has  the  same  fallacies,  but  is  said 
to  be  more  sensitive.  A  green  or  blue  color  shows  the  pres- 
ence of  hemoglobin. 

6.  Alkapton  Bodies. — The  name  "alkaptonuria"  has 
been  given  to  a  condition  in  which  the  urine  turns 
reddish  brown  to  brownish  black  upon  standing  and 
strongly  reduces  copper  (but  not  bismuth),  owing  to  the 
presence  of  certain  substances  which  result  from  imper- 
fect protein  metabolism.     The  change  of  color  takes 


158  THE   URINE 

place  quickly  when  fresh  urine  is  alkalinized,  hence  the 
name,  alkapton  bodies. 

Alkaptonuria  is  unaccompanied  by  other  symptoms, 
and  has  little  clinical  importance.  Only  about  forty-five 
cases,  mostly  congenital,  have  been  reported.  The 
change  in  color  of  the  urine  and  the  reduction  of  copper 
with  no  reduction  of  bismuth  nor  fermentation  with 
yeast  would  suggest  the  condition. 

7.  Melanin. — Urine  which  contains  melanin  likewise 
darkens  upon  exposure  to  the  air,  assuming  a  dark 
brown  or  black  color.  This  is  due  to  the  fact  that  the 
substance  is  eliminated  as  a  chromogen — melanogen — 
which  is  later  converted  into  the  pigment.  It  does  not 
reduce  copper. 

Melanuria  occurs  in  most,  but  not  all,  cases. of  mela- 
notic sarcoma.  Its  diagnostic  value  is  lessened  by  the 
fact  that  it  has  been  observed  in  other  wasting  diseases. 

Tests  for  Melanin, — (i)  Addition  of  ferric  chlorid  gives  a 
gray  precipitate  which  blackens  on  standing. 

(2)  Bromin  water  causes  a  yellow  precipitate  which 
gradually  turns  black. 

8.  Urobilin. — Traces  of  this  urinary  pigment  may 
be  present  under  normal  conditions.  It  is  generally 
regarded  as  identical  with  hydrobilirubin  of  the  feces. 
It  is  excreted  as  a  chromogen,  urobilinogen,  which  is 
changed  into  urobiUn  through  the  action  of  light  within 
a  few  hours  after  the  urine  is  voided.  A  great  excess 
usually  gives  the  urine  a  dark-brown  color,  suggesting 
the  presence  of  bile.  Small  amounts  may  cause  no  per- 
ceptible change  in  color.  It  is  derived  from  biUrubin, 
which  is  transformetl   through  bacterial  action  in  the 


CHEMIC  EXAMINATION  159 

intestine  to  urobilinogen.  Under  normal  conditions  this 
is  absorbed,  carried  to  the  liver,  and  there  reconverted 
into  bilirubin.  When  the  liver  cells  are  deranged,  the 
transformation  into  bilirubin  does  not  take  place  and 
urobilinogen  is  excreted  by  the  kidneys. 

The  presence  of  any  considerable  quantity  of  the 
pigment  or  the  chromogen  in  the  urine  is,  therefore,  a 
qualitative  test  of  the  functional  incapacity  of  the  liver. 
CUnically,  urobilin  is  found  in  a  great  variety  of  diseases 
associated  with  hepatic  derangement.  It  is  especially 
marked  in  cirrhosis  of  the  liver. 

(i)  Test  for  Urobilinogen. — To  a  few  cubic  centimeters 
of  the  urine  in  a  test-tube  add  a  few  crystals  of  dimethyl- 
amino-benzaldehyd  and  make  definitely  acid  with  hydro- 
chloric acid.  In  the  presence  of  pathologic  amounts  of  uro- 
bilinogen an  intense  red  color  appears.  Normal  amounts 
will  cause  the  red  color  only  when  the  urine  is  heated. 

(2)  Schlesinger's  Test  for  Urobilin. — ^To  about  5  c.c.  of 
the  urine  in  a  test-tube  add  a  few  drops  of  Lugol's  solution 
to  transform  the  chromogen  into  the  pigment.  Now  add  4  or 
5  c.c.  of  a  saturated  solution  of  zinc  chlorid  in  absolute 
alcohol  and  filter.  A  greenish  fluorescence,  best  seen  when 
the  tube  is  viewed  against  a  black  background  and  the  light 
is  concentrated  upon  it  with  a  lens,  shows  the  presence  of 
urobilin.  Bile-pigment,  if  present,  should  be  removed  by 
adding  about  one-fifth  volume  of  10  per  cent,  calcium  chlorid 
solution  and  filtering. 

9.  Diazo  Substances. — Certain  unknown  substances 
sometimes  present  in  the  urine  give  a  characteristic 
color  reaction — the  "diazo  reaction"  of  Ehrlich — when 
treated  with  diazo-benzol-sulphonic  acid  and  ammonia. 
This  reaction  has  much  cHnical  value,  provided  its  lim- 


l6o  THE   URINE 

itations  be  recognized.  It  is  at  best  an  empirical  test 
and  must  be  interpreted  in  the  light  of  cUnical  symptoms. 
Although  it  has  been  met  with  in  a  considerable  number 
of  diseases,  its  usefulness  is  practically  limited  to  ty- 
phoid fever,  tuberculosis,  and  measles. 

(i)  Typhoid  Fever. — Practically  all  cases  give  a 
positive  reaction,  which  varies  in  intensity  with  the 
severity  of  the  disease.  It  is  so  constantly  present  that 
it  is  sometimes  said  to  be  "negatively  pathognomonic": 
if  negative  upon  several  successive  days  at  a  stage  of  the 
disease  when  it  should  be  positive,  typhoid  is  almost 
certainly  absent.  Upon  the  other  hand,  a  reaction 
when  the  urine  is  highly  diluted  (i :  50  or  more)  has 
much  positive  diagnostic  value,  since  this  dilution  pre- 
vents the  reaction  in  most  conditions  which  might  be 
mistaken  for  typhoid;  but  it  should  be  noted  that  mild 
cases  of  t^^hoid  may  not  give  it  at  this  dilution.  Ordi- 
narily the  diazo-  appears  a  little  earlier  than  the  Widal 
reaction, — about  the  fourth  or  fifth  day, — but  it  may  be 
delayed.  In  contrast  to  the  Widal,  it  begins  to  fade 
about  the  end  of  the  second  week,  and  soon  thereafter 
entirely  disappears.  An  early  disappearance  is  a  favor- 
able sign.  It  reappears  during  a  relapse,  and  thus  helps 
to  distinguish  between  a  relapse  and  a  complication,  in 
which  it  does  not  reappear. 

(2)  Tuberculosis. — The  diazo-reaction  has  been  ob- 
tained in  many  forms  of  the  disease.  It  has  little  or 
no  diagnostic  value.  Its  continued  presence  in  pul- 
monary tuberculosis  is,  however,  a  grave  prognostic 
sign,  even  when  the  physical  signs  are  slight.  After  it 
once  appears  it  generally  persists  more  or  less  intermit- 
tently until  death,  the  average  length  of  Ufe  after  its 


CHEMIC   EXAMINATION  l6l 

appearance  being  about  six  months.  The  reaction  is 
often  temporarily  present  in  mild  cases  during  febrile 
complications,  and  has  then  no  significance. 

(3)  Measles. — A  positive  reaction  is  usually  obtained 
in  measles,  and  may  help  to  distinguish  this  disease 
from  German  measles,  in  which  it  does  not  occur.  It 
generally  appears  before  the  eruption  and  remains  about 
five  days. 

Technic. — Although  the  test  is  really  a  very  simple  one, 
careful  attention  to  technic  is  imperative.  Many  of  the  early 
workers  were  very  lax  in  this  regard.  Faulty  technic  and 
failure  to  record  the  stage  of  the  disease  in  which  the  tests 
were  made  have  probably  been  responsible  for  the  bulk  of 
the  conflicting  results  reported. 

Certain  drugs  often  given  in  tuberculosis  and  typhoid 
interfere  with  or  prevent  the  reaction.  The  chief  are  creosote, 
tannic  acid  and  its  compounds,  opium  and  its  alkaloids,  salol, 
phenol,  and  the  iodids.     The  reagents  are: 

(i)  Saturated  solution  sulphanilic  acid  in  5  per  cent, 
hydrochloric  acid. 

(2)  Aqueous  solution  sodium  nitrite  (0.5  per  cent.). 

(3)  Strong  ammonia. 

Mix  100  parts  of  (i)  and  i  part  of  (2).^  In  a  test-tube 
take  equal  parts  of  this  mixture  and  the  iu"ine,  and  pour  i  or 
2  c.c.  of  the  ammonia  upon  its  surface.  If  the  reaction  be 
positive,  a  garnet  ring  will  form  at  the  junction  of  the  two 
fluids;  and,  upon  shaking,  a  distinct  pink  color  will  be  im- 
parted to  the  foam.  The  color  of  the  foam  is  the  essential 
feature.  If  desired,  the  mixture  may  be  well  shaken  before 
the  ammonia  is  added:  the  pink  color  will  then  instantly 
appear  in  that  portion  of  the  foam  which  the  ammonia  has 

^  These  proportions  are  recommended  by  Greene,  and  are  now  gen- 
erally used.      Ehrlich  used  40  parts  of  (i)  and  i  part  of  (2). 
11 


l62  THE    URINE 

reached,  and  can  be  readily  seen.  The  color  varies  from  eosin- 
pink  to  deep  crimson,  dei)ending  ujx)n  the  intensity  of  the 
reaction.  //  is  a  pure  pink  or  red;  any  trace  of  yellow  or  orange 
denotes  a  negative  reaction.  A  doubtful  reaction  should  be 
considered  negative. 

Substitutes  for  the  Diazo-reaction. — Two  tests,  which 
have  been  offered  as  simple  and  satisfactory  substitutes 
for  the  diazo,  have  recently  found  rather  wide  accept- 
ance. They  are  supposed  to  be  positive  in  the  same 
classes  of  cases  as  the  diazo  and  to  have  the  same  clin- 
ical significance,  but  are  claimed  to  be  more  reliable. 

(i)  Russo's  Methylene-blue  Test. — To  5  c.c.  of  the  urine 
in  a  test-tube  add  5  drops  of  i  :  1000  aqueous  solution  of 
methylene-blue,  and  mix.  An  emerald  or  mint-green  color, 
in  which  there  must  be  no  trace  of  blue,  denotes  a  positive 
reaction.  There  is  considerable  difficulty  in  judging  the 
color.  The  correct  color  may  be  obtained  for  comparison 
by  adding  the  methylene-blue  solution  to  a  bile-tinged  urine. 

Since  this  test  was  offered  in  1905,  it  has  been  condemned 
by  many  workers  and  e.x tolled  by  others.  The  writer's  expe- 
rience with  it  has  been  unfavorable. 

(2)  Weisz's  Permanganate  Test. — In  a  test-tube  mix  i  c.c. 
of  urine  and  2  c.c.  distilled  water,  and  add  3  drops  of  i  :  1000 
aqueous  solution  of  potassium  permanganate.  The  appear- 
ance of  a  yellow  color  denotes  a  positive  reaction.  As  sug- 
gested by  Heflebower,  the  color  is  best  judged  by  comparison 
with  a  tube  of  diluted  urine  to  which  no  permanganate  has 
been  added. 

Weisz  believes  the  diazo-reaction  to  be  due  principally  to 
urochromogen,  which,  because  of  the  effect  of  certain  toxins 
upon  metabolism,  fails  of  conversion  into  urochrome;  and  he 
has  offered  (191 1)  the  permanganate  reaction  as  a  more  satis- 


CHEMIC  EXAMINATION  1 63 

factory  test,  both  for  urochromogen  and  for  an  antecedent 
substance  which  has  the  same  significance  as  urochromogen, 
but  which  the  diazo  fails  to  detect.  This  test  has  been 
studied  chiefly  in  its  relation  to  prognosis  in  tuberculosis,  in 
which  it  appears  to  have  about  the  same  value  as  the  diazo, 
with  the  advantage  that  it  is  more  frequently  noted  and  is 
less  intermittent  in  a  given  case. 

10.  Pancreatic  Reaction. — Cammidge  has  shown  that 
in  cases  of  pancreatitis  a  substance  capable  of  forming 
crystals  with  phenylhydrazin  can  be  developed  by 
boiling  the  urine  with  a  mineral  acid,  and  has  offered  the 
following  test  as  an  aid  in  diagnosis  of  this  obscure  con- 
dition. The  nature  both  of  this  substance  and  the  ante- 
cedent substance  from  which  it  is  derived  is  not  known. 
The  difl&culty  and  importance  of  diagnosis  in  pancreatitis 
warrant  inclusion  of  the  method  here,  even  though  more 
recent  work  indicates  that  its  value  is  by  no  means  so 
great  as  originally  claimed. 

While  the  test  is  somewhat  tedious,  all  the  manipula- 
tions are  simple  and  require  no  apparatus  but  flasks, 
test-tubes,  and  funnels. 

Technic. — Careful  attention  to  detail  is  imperative.  An 
ordinary  routine  examination  is  first  made.  Albimiin  and 
sugar,  if  present,  must  be  removed:  the  former,  by  acidifying 
with  acetic  acid,  boiling,  and  filtering;  the  latter,  by  fermenta- 
tion with  yeast  after  the  first  step  of  the  method  proper.  An 
alkaline  urine  should  be  made  slightly  acid  with  hydrochloric 
acid. 

(i)  Forty  cubic  centimeters  of  the  urine,  which  has  been 

.rendered  perfectly  clear  by  repeated  filtration  through  the 

same  filter-paper,  are  placed  in  a  small  flask,  treated  with  i 

c.c.  concentrated  hydrochloric  acid  and  gently  boiled  on  a 


i64 


THE  UIONE 


sand-bath  for  ten  minutes,  a  funnel  with  long  stem  being 
placed  in  the  neck  of  the  flask  to  act  as  a  condenser  (Fig.  34). 
After  boiling,  the  urine  is  cooled  in  a  stream  of  cold  water 
and  brought  to  its  original  bulk  with  distilled  water;  8  gm.  of 
lead  carbonate  are  then  added  to  neutralize  the  acid.  The 
fluid  is  allowed  to  stand  a  few  minutes  and  then  filtered 
through  well-moistened  fine-grain  filter-paper  imtil  perfectly 
clear. 


Fig.  34- 


"Pancreatic  reaction"  ilasks  liitftl  wiih  funnel  condensers  on  a  sand-bath 
(Robson  and  Cammidge). 


(2)  The  filtrate  is  shaken  up  with  8  gm.  powdered  tribasic 
lead  acetate  and  filtered.  The  excess  of  lead  is  then  removed 
by  passing  hydrogen  sulphid  gas  through  the  fluid  (see  page 
170)  or  by  shaking  well  with  4  gm.  finely  powdered  sodium 
sulphate,  heating  to  boiling,  cooling  to  as  low  a  temperature 
as  possible  in  a  stream  of  water,  and  filtering  as  before  imtil 
p>erfectly  clear. 


CHEMIC   EXAMINATION 


165 


(3)  Ten  cubic  centimeters  of  the  filtrate  are  then  made 
up  to  17  c.c.  with  distilled  water,  and  added  to  a  mixture  of 
0.8  gm.  phenylhydrazin  hydrochlorate,  2  gm.  powdered  so- 
dium acetate,  and  i  c.c,  50  per  cent,  acetic  acid  in  a  small  flask 
with  funnel  condenser.  This  is  boiled  on  a  sand-bath  for  ten 
minutes,  and  filtered  while  hot  through  filter-paper  moistened 


Fig.  3S- — Improved  "pancreatic  reaction."  Crystals  obtained  from  a  case  of  chronic 
pancreatitis  with  gall-stones  in  the  common  duct  ( X  200)  (from  a  photo  by  P.  J.  Cam- 
midge). 


with  hot  water  into  a  test-tube  with  a  15  c.c.  mark.  Should 
the  filtrate  not  reach  this  mark,  make  up  to  15  c.c.  with  hot 
distilled  water.    Allow  to  cool  slowly. 

(4)  In  well-marked  cases  of  pancreatitis  a  yellow  pre- 
cipitate appears  within  a  few  hours ;  in  milder  cases  it  may 
not  appear  for  twelve  hours.  The  microscope  shows  this 
sediment  to  consist  of  ''long,  light  yellow,  flexible,  hair-like 


1 66  THE   URINE 

crystals  arranged  in  sheaves,  which,  when  irrigated  with  33 
per  cent,  sulphuric  acid,  melt  away  and  disappear  in  ten  to 
fifteen  seconds  after  the  acid  first  touches  them"  (Fig.  35). 
(5)  To  exclude  traces  of  glucose  which  might  be  overlooked 
in  the  preliminary  examination  a  control  test  should  be  carried 
out  in  the  same  manner,  with  omission  of  Step  (i). 


1 1 .  Drugs. — The  effect  of  various  drugs  upon  the 
color  of  the  urine  has  been  mentioned  (see  p.  89).  Most 
poisons  are  eliminated  in  the  urine,  but  their  detection 
is  more  properly  discussed  in  works  upon  toxicology.  A 
few  drugs  which  are  of  interest  to  the  practitioner,  and 
which  can  be  detected  by  comparatively  simple  methods, 
are  mentioned  here. 

Acetanilid  and  Phenacetin. — The  urine  is  evaporated 
by  gentle  heat  to  about  half  its  volume,  boiled  for  a  few 
minutes  with  about  one-fifth  its  volume  of  strong  hydro- 
chloric acid,  'and  shaken  out  with  ether.  The  ether  is 
evaporated,  the  residue  dissolved  in  water,  and  the 
following  test  applied:  To  about  10  c.c.  are  added  a  few 
cubic  centimeters  of  3  per  cent,  phenol,  followed  by  a 
weak  solution  of  chromium  trioxid  (chromic  acid)  drop 
by  drop.  The  fluid  assumes  a  red  color,  which  changes 
to  blue  when  ammonia  is  added.  If  the  urine  is  very 
pale,  extraction  with  ether  may  be  omitted. 

Antipyrin. — This  drug  gives  a  dark-red  color  when  a 
few  drops  of  10  per  cent,  ferric  chlorid  are  added  to  the 
urine.  The  color  does  not  disappear  upon  boiling,  which 
excludes  diacetic  acid. 

Arsenic. — Reinsch^s  Test. — Add  to  the  urine  in  a  test- 
tube  or  small  flask  about  one-seventh  its  volume  of  hy- 
drochloric acid,  introduce  a  piece  of  bright  copper-foil 


CHEMIC  EXAMINATION  167 

about  I  inch  square,  and  boil  for  several  minutes. 
If  arsenic  be  present,  a  dark-gray  film  is  deposited 
upon  the  copper.  The  test  is  more  deUcate  if  the  urine 
be  concentrated  by  slow  evaporation.  This  test  is  well 
known  and  is  widely  used,  but  is  not  so  reliable  as  the 
following : 

GutzeWs  Test. — In  a  large  test-tube  place  a  little 
arsenic-free  zinc,  and  add  5  to  10  c.c.  pure  dilute  hydro- 
chloric acid  and  a  few  drops  of  iodin  solution  (Gram's 
solution  will  answer),  then  add  5  to  10  c.c.  of  the  urine. 
At  once  cover  the  mouth  of  the  tube  with  a  filter-paper 
cap  moistened  with  saturated  aqueous  solution  of  silver 
nitrate  (1:1)  If  arsenic  be  present,  the  paper  quickly 
becomes  lemon  yellow,  owing  to  formation  of  a  com- 
pound of  silver  arsenid  and  silver  nitrate,  and  turns  black 
when  touched  with  a  drop  of  water.  To  make  sure  that 
the  reagents  are  arsenic  free,  the  paper  cap  may  be  ap- 
plied for  a  few  minutes  before  the  urine  is  added. 

Atropin  will  cause  dilatation  of  the  pupil  when  a  few 
drops  of  the  urine  are  placed  in  the  eye  of  a  cat  or  rabbit. 

Bromids  can  be  detected  by  acidifying  about  10  c.c.  of 
the  urine  with  dilute  sulphuric  acid,  adding  a  few  drops 
of  faming  nitric  acid  and  a  few  cubic  centimeters  of 
chloroform,  and  shaking.  In  the  presence  of  bromin  the 
chloroform,  which  settles  to  the  bottom,  assumes  a  yellow 
color. 

Chloral  hydrate  appears  in  the  urine  chiefly  as  uro- 
chloralic  acid,  which  reduces  the  copper  solutions  used  for 
sugar  tests.  To  detect  it,  evaporate  about  500  c.c.  of  the 
-urine  to  about  one-fourth  its  volume,  make  decidedly 
acid  with  hydrochloric  acid,  add  about  50  c.c.  of  ether, 
shake  thoroughly,  and  separate  the  ether.    Now  evapor- 


l68  THE  URINE 

ate  the  ether  and  dissolve  the  residue  in  a  little  water. 
If  urochloralic  acid  be  present  this  aqueous  solution 
will  respond  to  Fehling's  test. 

Hexamethylenamin. — ^Interest  in  this  drug  centers 
chiefly  in  its  value  as  a  urinary  antiseptic,  which  depends 
upon  its  decomposition  with  liberation  of  formaldehyd. 
According  to  a  number  of  recent  workers  formaldehyd 
can  be  detected  in  the  urine  of  only  about  50  per  cent, 
of  patients  who  are  taking  hexamethylenamin.  A  test 
for  formaldehyd  is,  therefore,  necessary  in  order  to 
know  whether  the  object  in  administering  the  drug  is 
being  accompUshed. 

Rimini-Bumam  Test  for  Formaldehyd. — To  about  10  c.c. 
of  the  urine  add  successively  3  drops  of  0.5  per  cent,  solution 
of  phenylhydrazin  hydrochlorid,  3  drops  of  5  per  cent,  solu- 
tion of  sodium  nitroprussid,  and  a  few  drops  of  a  saturated 
solution  of  sodium  hydroxid.  The  last  is  allowed  to  trickle 
down  the  inside  of  the  tube;  and  if  formaldehyd  be  present 
a  purplish-black  color,  changing  to  green  and  then  to  yellow, 
will  appear  as  it  mingles  with  the  luine. 

lodin  from  ingestion  of  iodids  or  absorption  from 
iodoform  dressings  is  tested  for  in  the  same  way  as  the 
bromids,  the  chloroform  assuming  a  pink  to  reddish- 
violet  color;  or  Obermayer's  reagent  may  be  used  in  the 
same  way  as  described  for  indican  (see  p.  113).  To  de- 
tect traces,  a  large  quantity  of  urine  should  be  rendered 
alkaline  with  sodium  carbonate  and  greatly  concen- 
trated by  evaporation  before  testing. 

Lead. — No  simple  method  is  sufficiently  sensitive  to 
detect  the  traces  of  lead  which  occur  in  the  urine  in 
chronic  poisoning.    Of  the  more  sensitive  methods,  that 


CHEMIC   EXAMINATION  1 69 

of  Arthur  Lederer  is  probably  best  suited  to  the  prac- 
titioner : 

It  is  essential  that  all  apparatus  used  be  lead  free. 
Five  hundred  cubic  centimeters  of  the  urine  are  acidified 
with  70  c.c.  pure  sulphuric  acid,  and  heated  in  a  beaker 
or  porcelain  dish.  About  20  to  25  gm.  of  potassium 
persulphate  are  added  a  little  at  a  time.  This  should 
decolorize  the  urine,  leaving  it  only  slightly  yellow.  If 
it  darkens  upon  heating,  a  few  more  crystals  of  potassium 
persulphate  are  added,  the  burner  being  first  removed  to 
prevent  boiling  over;  if  it  becomes  cloudy,  a  small  amount 
of  sulphuric  acid  is  added.  It  is  then  boiled  until  it  has 
evaporated  to  250  c.c.  or  less.  After  cooling,  an  equal 
volume  of  alcohol  is  added,  and  the  mixture  allowed  to 
stand  in  a  cool  place  for  four  or  five  hours,  during  which 
time  all  the  lead  will  be  precipitated  as  insoluble  sulphate. 

The  mixture  is  then  filtered  through  a  small,  close- 
grained  filter-paper  (preferably  an  ashless,  quantitative 
filter-paper),  and  any  sediment  remaining  in  the  beaker 
or  dish  is  carefully  washed  out  with  alcohol  and  filtered. 
A  test-tube  is  placed  underneath  the  funnel;  a  hole  is 
punched  through  the  tip  of  the  filter  with  a  small  glass 
rod,  and  all  the  precipitate  (which  may  be  so  slight  as  to 
be  scarcely  visible)  washed  down  into  the  test-tube  with  a 
jet  of  distilled  water  from  a  wash-bottle,  using  as  httle 
water  as  possible.  Ten  cubic  centimeters  will  usually 
suffice.  This  fluid  is  then  heated,  adding  crystals  of 
sodium  acetate  until  it  becomes  perfectly  clear.  It  now 
contains  all  the  lead  of  the  500  c.c.  urine  in  the  form  of 
lead  acetate.  It  is  allowed  to  cool,  and  hydrogen  sulphid 
gas  is  passed  through  it  for  about  five  minutes.  The 
slightest    yellowish-brown    discoloration   indicates    the 


1 70  THE   URINE 

presence  of  lead.  A  very  slight  discoloration  can  be 
best  seen  when  looked  at  from  above.  For  comparison, 
the  gas  may  be  passed  through  a  test-tube  containing 
an  equal  amount  of  distilled  water.  The  quantity  of 
lead  can  be  determined  by  comparing  the  discoloration 
with  that  produced  by  passing  the  gas  through  lead 
acetate  (sugar  of  lead)  solutions  of  known  strength. 
One  gram  of  lead  acetate  crystals  contains  0.54  gram  of 
lead.    Hydrogen  sulphid  is  easily  prepared  in  the  simple 


Fig.  36. — A  simple  liydrogen  sulphid  generator. 

apparatus  shown  in  Fig.  36.  A  small  quantity  of  iron 
sulphid  is  placed  in  the  test-tube ;  a  little  dilute  hydro- 
chloric acid  is  added ;  the  cork  is  replaced ;  and  the  dehvery 
tube  is  inserted  to  the  bottom  of  the  fluid  to  be  tested. 

Mercury. — Traces  can  be  detected  in  the  urine  for  a 
considerable  time  after  the  use  of  mercury  compounds 
by  ingestion  or  inunction. 

About  a  liter  of  urine  is  acidified  with  10  c.c.  hydro- 
chloric acid,  and  a  small  piece  of  copper-foil  or  gauze  is 


CHEMIC   EXAMINATION  17I 

introduced.  This  is  gently  heated  for  an  hour,  and 
allowed  to  stand  for  twenty-four  hours.  The  metal  is 
then  removed,  and  washed  successively  with  very  dilute 
sodium  hydroxid  solution,  alcohol,  and  ether.  When 
dry,  it  is  placed  in  a  long,  slender  test-tube,  and  the 
lower  portion  of  the  tube  is  heated  to  redness.  A  tube 
with  a  constriction  in  its  upper  portion  is  better.  If 
mercury  be  present,  it  will  volatilize  and  condense  in 
the  upper  portion  of  the  tube  as  small,  shining  globules 
which  can  be  Seen  with  a  hand-magnifier  or  low  power 
of  the  microscope.  If,  now,  a  crystal  of  iodin  be  dropped 
into  the  tube  and  gently  heated,  the  mercury  upon  the 
side  of  the  tube  is  changed  first  to  the  yellow  iodid,  and 
later  to  the  red  iodid,  which  are  recognized  by  their  color. 

Morphin. — Add  sufficient  ammonia  to  the  urine  to 
render  it  distinctly  ammoniacal,  and  shake  thoroughly 
with  a  considerable  quantity  of  pure  acetic  ether.  Sepa- 
rate the  ether  and  evaporate  to  dryness.  To  a  little  of 
the  residue  in  a  watch-glass  or  porcelain  dish  add  a  few 
drops  of  formaldehyd-sulphuric  acid,  which  has  been 
freshly  prepared  by  adding  i  drop  of  formalin  to  i  c.c. 
pure  concentrated  sulphuric  acid.  If  morphin  be  pres- 
ent, this  will  produce  a  purple-red  color,  which  changes 
to  violet,  blue  violet,  and  finally  nearly  pure  blue. 

Phenol. — As  has  been  stated,  the  urine  following 
phenol-poisoning  turns  olive  green  and  then  brownish 
black  upon  standing.  Tests  are  of  value  in  recognizing 
poisoning  from  ingestion  and  in  detecting  absorption 
from  carboHzed  dressings. 

-  The  urine  is  acidulated  with  hydrochloric  acid  and 
distilled.  To  the  first  few  cubic  centimeters  of  distillate 
is  added  10  per  cent,  solution  of  ferric  chlorid  drop  by 


172  THE  ITRINE 

drop.  The  presence  of  phenol  causes  a  deep  amethyst- 
blue  color,  as  in  Uffclmann's  test  for  lactic  acid. 

Phenolphthalein,  which  is  now  widely  used  as  a  ca- 
thartic, gives  a  bright  pink  color  when  the  urine  is  ren- 
dered alkaline. 

Quinin. — A  considerable  quantity  of  the  urine  is  ren- 
dered alkaline  with  ammonia  and  extracted  with  ether; 
the  ether  is  evaporated,  and  a  portion  of  the  residue 
dissolved  in  about  20  drops  of  dilute  alcohol.  The 
alcoholic  solution  is  acidulated  with  dilute  sulphuric 
acid,  I  drop  of  an  alcoholic  solution  of  iodin  (tincture 
of  iodin  diluted  about  ten  times)  is  added,  and  the  mix- 
ture is  warmed.  Upon  cooling,  an  iodin  compound  of 
quinin  (herapathite)  will  separate  out  in  the  form  of  a 
microcrystalline  sediment  of  green  plates. 

The  remainder  of  the  residue  may  be  dissolved  in  a 
little  dilute  sulphuric  acid.  This  solution  will  show  a 
characteristic  blue  fluorescence  when  quinin  is  present. 

Resinous  drugs  cause  a  white  precipitate  like  that  of 
albumin  when  strong  nitric  acid  is  added  to  the  urine. 
This  is  dissolved  by  alcohol. 

Salicylates,  salol,  aspirin,  and  similar  drugs  give  a 
bluish-violet  color,  which  does  not  disappear  upon  heat- 
ing, upon  addition  of  a  few  drops  of  10  per  cent,  ferric 
chlorid  solution.  When  the  quantity  of  salicylates  is 
small,  the  urine  may  be  acidified  with  hydrochloric  acid 
and  extracted  with  ether,  the  ether  evaporated,  and  the 
test  applied  to  an  aqueous  solution  of  the  residue. 

Tannin  and  its  compounds  appear  in  the  urine  as 
gallic  acid,  and  the  urine  becomes  greenish  black  (inky, 
if  much  gallic  acid  be  present)  when  treated  with  a  solu- 
tion of  ferric  chlorid. 


MICROSCOPIC   EXAMINATION  1 73 

nL  MICROSCOPIC  EXAMINATION 

A  careful  microscopic  examination  will  often  reveal 
structures  of  great  diagnostic  importance  in  urine  which 
seems  perfectly  clear,  and  from  which  only  very  slight 
sediment  can  be  obtained  with  the  centrifuge.  Upon  the 
other  hand,  cloudy  urines  with  abundant  sediment  are 
often  shown  by  the  microscope  to  contain  nothing  of 
clinical  significance. 

Since  the  nature  of  the  sediment  soon  changes,  the 
urine  must  be  examined  while  fresh,  preferably  within  six 
hours  after  it  is  voided.  When  possible  it  should  be 
kept  on  ice.  The  sediment  is  best  obtained  by  means  of 
the  centrifuge.  If  a  centrifuge  is  not  available,  the  urine 
may  be  allowed  to  stand  in  a  conical  test-glass  for  six 
to  twenty-four  hours  after  adding  some  preservative 
(seep.  86). 

A  small  amount  of  the  sediment  should  be  transferred 
to  a  slide  by  means  of  a  pipet.  It  is  very  important  to  do 
this  properly.  The  best  pipet  is  a  small  glass  tube  which 
has  been  drawn  out  at  one  end  to  a  tip  with  rather  small 
opening.  The  tube  or  glass  containing  the  sediment  is 
held  on  a  level  with  the  eye,  the  larger  end  of  the  pipet  is 
closed  with  the  index-finger,  which  must  be  dry,  and  the 
tip  is  carried  down  into  the  sediment.  By  carefully 
loosening  the  finger,  but  not  entirely  removing  it,  a  small 
amount  of  the  sediment  is  then  allowed  to  run  slowly  into 
the  pipet.  Slightly  rotating  the  pipet  will  aid  in  accom- 
plishing this,  and  at  the  same  time  will  serve  to  loosen 
any  structures  which  cling  to  the  bottom  of  the  tube. 
After  wiping  off  the  urine  which  adheres  to  the  outside, 
a  drop  from  the  pipet  is  placed  upon  a  clean  slide.  A 
hair  is  then  placed  in  the  drop  and  a  large  cover-glass 


174  THE   URINE 

applied.  The  correct  size  of  the  drop  can  be  learned 
only  by  experience.  It  should  not  be  so  large  as  to  float 
the  cover-glass  about,  nor  so  small  as  to  leave  unoccu- 
pied space  beneath  the  cover.  Many  workers  use  no 
cover.  This  offers  a  thicker  layer  and  larger  area  of 
urine,  the  chance  of  finding  scanty  structures  being 
proportionately  increased.  It  has  the  disadvantage  that 
any  jarring  of  the  room  (as  by  persons  walking  about) 
sets  the  microscopic  field  into  vibratory  motion  and 
makes  it  impossible  to  see  anything  clearly;  and,  since  it 
does  not  allow  of  the  use  of  high-power  objectives,  one 
cannot  examine  details  as  one  often  wishes  to  do.  It  is 
true  that  a  cover  can  be  applied  later,  but  any  structure 
which  one  has  found  with  the  low  power  and  wishes  to 
study  with  the  high  is  sure  to  be  lost  when  the  cover  is 
applied.  A  large  cover-glass  (about  22  mm.  square) 
with  a  hair  beneath  it  avoids  these  disadvantages,  and 
gives  enough  urine  to  find  any  structures  which  are 
present  in  sufficient  number  to  have  clinical  significance, 
provided  other  points  in  the  technic  have  been  right. 
It  is  best,  however,  to  examine  several  drops;  and,  when 
the  sediment  is  abundant,  drops  from  the  upper  and  lower 
portions  should  be  examined  separately. 

In  examining  urinary  sediments  microscopically  no 
fault  is  so  common,  nor  so  fatal  to  good  results,  as  im- 
proper illumination  (see  Fig.  4),  and  none  is  so  easily 
corrected.  The  light  should  be  central  and  very  sub- 
dued for  ordinary  work,  but  oblique  illumination,  ob- 
tained by  swinging  the  mirror  a  little  out  of  the  optical 
axis,  will  be  found  helpful  in  identifying  certain  delicate 
structures  like  hyaline  casts.  The  i6-mm.  objective 
should  be  used  as  a  finder,  while  the  4-mm.  is  reserved 


MICROSCOPIC   EXAMINATION  1 75 

for  examining  details.  An  experienced  worker  will  rely 
almost  wholly  upon  the  lower  power. 

It  is  well  to  emphasize  that  the  most  common  errors 
which  result  in  failure  to  find  important  structures,  when 
present,  are:  (a)  lack  of  care  in  transferring  the  sediment  to 
the  slide,  (b)  too  strong  illumination,  and  (c)  too  great  mag- 
nification. 

In  order  to  distinguish  between  similar  structures  it  is 
often  necessary  to  watch  the  effect  upon  them  of  certain 
reagents.  This  is  especially  true  of  the  various  unorgan- 
ized sediments.  They  very  frequently  cannot  be  identi- 
fied from  their  form  alone.  With  the  structures  still  in 
focus,  a  drop  of  the  reagent  may  be  placed  at  one  edge  of 
the  cover-glass  and  drawn  underneath  it  by  the  suction  of 
a  piece  of  blotting-paper  touched  to  the  opposite  edge; 
or  a  small  drop  of  the  reagent  and  of  the  urine  may  be 
placed  close  together  upon  a  slide  and  a  cover  gently 
lowered  over  them.  As  the  two  fluids  mingle,  the  effect 
upon  various  structures  may  be  seen. 

Urinary  sediments  may  be  studied  under  three  heads: 
A.  Unorganized  sediments.  B.  Organized  sediments.  C. 
Extraneous  structures. 

A.   Unorganized  Sediments 

In  general,  these  have  little  diagnostic  or  prognostic 
significance.  Most  of  them  are  substances  normally 
present  in  solution,  which  have  been  precipitated  either 
because  present  in  excessive  amounts,  or,  more  fre- 
quently, because  of  some  alteration  in  the  urine  (as  in 
reaction,  concentration,  etc.)  which  may  be  purely 
physiologic,  depending  upon  changes  in  diet  or  habits. 
Various  substances  are  always  precipitated  during  de- 


176 


THE   URINE 


composition,  which  may  take  place  either  within  or 
without  the  body.  Unorganized  sediments  may  be 
classified  according  to  the  reaction  of  the  urine  in  which 
they  are  most  likely  to  be  found.  This  classification  is 
useful,  but  is  not  accurate,  since  the  characteristic  sedi- 
ments of  acid  urine  may  remain  after  the  urine  has 
become  alkaline,  while  the  alkaline  sediments  may  be 
precipitated  in  a  urine  which  is  still  acid. 

In  acid  urine:  Uric  acid,  amorphous  urates,  sodium 
urate,  calcium  oxalate,  leucin  and  tyrosin,  cystin,  and 


f 


t 


Fig.  37. — Unusual  urinary  crystals  (drawn  from  various  authors):  i,  Calcium  sul- 
phate (colorless);  2,  cholesterin  (colorless);  3,  hippuric  acid  (colorless);  4,  hematoidin 
(brown);  5,  fatty  acids  (colorless);  6,  indigo  (blue);  7,  sodium  urate  (yellowish). 

fat-globules.  Uric  acid,  the  urates,  and  calcium  oxalate 
are  the  common  deposits  of  acid  urines;  the  others  are 
less  frequent,  and  depend  less  upon  the  reaction  of  the 
urine. 

In  alkaline  urine:  Phosphates,  calcium  carbonate,  and 
ammonium  urate. 

Other  crystalline  sediments  (Fig.  37)  which  are  rare 
and  require  no  further  mention  are:  Calcium  sulphate, 
cholesterin,  hippuric  acid,  hematoidin,  fatty  acids,  and 
indigo. 


MICROSCOPIC   EXAMINATION  1 77 

The  following  brief  table  will  aid  the  student  in  identi- 
fying the  chemical  sediments  which  one  meets  every 
day: 

Acid  urine.  Alkaline  urine. 

Yellow  crystals.  Uric  acid — dissolve  in    Ammonium      urate — dis- 

KOH.  solve  in  HCl. 

Colorless  crystals.        Calcium  oxalate — dis-    Phosphate     crystals — dis- 
solve in  HCl  solve  in  acetic  acid. 

Amorphous  material.  Urates — dissolve  with    Amorphous  phosphates — 
heat.  dissolve  in  acetic  acid. 

1.  In  Acid  Urine. — (i)  Uric-acid  Crystals. — These 
crystals  are  the  red  grains — "gravel"  or  "red  sand" — 
which  are  often  seen  adhering  to  the  sides  and  bottom 
of  a  vessel  containing  urine.  Microscopically,  they  are 
yellow  or  reddish-brown  crystals,  which  differ  greatly  in 
size  and  shape.  The  color  is  due  to  urinary  pigments. 
The  most  characteristic  forms  (Plate  IV  and  Fig.  38,  2) 
are  "whetstones";  roset-like  clusters  of  prisms  and  whet- 
stones; and  rhombic  plates,  which  have  usually  a  paler 
color  than  the  other  forms  and  are  sometimes  colorless. 
A  very  rare  form  is  a  colorless  hexagonal  plate  resem- 
bling cysrin.  Recognition  of  the  crystals  depends  less 
upon  their  shape  than  upon  their  color,  the  reaction  of 
the  urine,  and  the  facts  that  they  are  soluble  in  caustic 
soda  solution  and  insoluble  in  hydrochloric  or  acetic  acid. 
When  ammonia  is  added,  they  dissolve  and  crystals  of 
ammonium  urate  appear. 

A  deposit  of  uric-acid  crystals  has  no  significance  un- 
less it  occurs  before  or  very  soon  after  the  urine  is  voided. 
Every  urine,  if  kept  acid,  will  in  time  deposit  its  uric 
acid.  Factors  which  favor  an  early  deposit  are  high 
acidity,  diminished  urinary  pigments,  and  excessive  ex- 
12 


178 


THE   URINE 


crction  of  uric  acid.  The  chief  clinical  interest  of  the 
crystals  lies  in  their  tendency  to  form  calcuU,  owing  to 
the  readiness  with  which  they  collect  about  any  solid 
object.  Their  presence  in  the  freshly  voided  urine  in 
clusters  of  crystals  suggests  stone  in  the  kidney  or  blad- 
der, especially  if  blood  is  also  present  (see  Fig.  65). 


Fig.  38. — Forms  of  uric  acid:  i,  Rhombic  plates;  2,  whetstone  forms;  3.  3,  quadrate 
forms;  4.  s,  prolonged  into  points;  6,  8,  rosets";  7,  pointed  bundles;  8,  barrel  forms  pre- 
cipitated by  adding  hydriKhloric  acid  to  urine  (Ogden). 


(2)  Amorphous  Urates. — These  are  chiefly  urates  of 
sodium  and  potassium  which  are  thrown  out  of  solution 
as  a  yellow  or  red  "brick-dust"  deposit.  In  pale  urines 
this  sediment  is  almost  white.  It  disappears  upon 
heating.  A  deposit  of  amorphous  urates  is  very  common 
in  concentrated  and  strongly  acid  urines,  especially  in 
cold  weather,  and  has  no  clinical  significance.     Under 


PLATE  IV 


V--/-V    ■•■■ 


rc 


(?■  "-? 


d 


'^^^ 


n 


,  / 


^  ;„*#■■;.«  s%^^  ■'■.■■■■■ 


Uric-acid   crystals  with  amorphous  urates  (after  Peyer). 


MICROSCOPIC   EXAMINATION  1 79 

the  microscope  it  appears  as  fine  yellowish  granules, 
often  so  abundant  as  to  obscure  all  other  structures 
(Plate  IV).  In  such  cases  the  urine  should  be  warmed 
before  examining.  Amorphous  urates  are  readily  sol- 
uble in  caustic  soda  solutions.  When  treated  with  hy- 
drochloric or  acetic  acid,  they  slowly  dissolve  and  rhom- 
bic crystals  of  uric  acid  appear. 

Rarely,  sodium  urate  occurs  in  crystalline  form — 
slender  prisms,  arranged  in  fan-  or  sheaf-like  structures 
(see  Fig.  37). 


0  o         ^ 

Fig.  39. — Various  forms  of  calcium  oxalate  crystals  (Ogden). 

(3)  Calcium  Oxalate. — Characteristic  of  calcium  oxa- 
late are  colorless,  glistening,  octahedral  crystals,  giving 
the  appearance  of  small  squares  crossed  by  two  intersect- 
ing diagonal  lines — the  so-called  "envelope  crystals" 
(see  Fig.  52).  They  vary  greatly  in  size,  being  sometimes 
so  small  as  to  seem  mere  points  of  light  with  medium- 
power  objectives.  Unusual  forms,  which,  however, 
seldom  occur  except  in  conjunction  with  the  octahedra, 
are  colorless  dumb-bells,  spheres,  and  variations  of  the 
octahedra  (Fig.  3q).  The  spheres  might  be  mistaken  for 
globules   of  fat   or  red  blood-corpuscles.     Crystals   of 


l8o  THE   URINE 

calcium  oxalate  are  insoluble  in  acetic  acid  or  caustic 
soda.  They  are  dissolved  by  strong  hydrochloric  acid, 
and  recrystallize  as  octahedra  upon  addition  of  ammonia. 
They  are  sometimes  encountered  in  alkaline  urine. 

The  crystals  are  commonly  found  in  the  urine  after 
ingestion  of  vegetables  rich  in  oxaHc  acid,  as  tomatoes, 
spinach,  asparagus,  and  rhubarb.  They  have  no  defi- 
nite significance  pathologically.  They  often  appear  in 
digestive  disturbances,  in  neurasthenia,  and  when  the 
oxidizing  power  of  the  system  is  diminished.  When 
abundant,  they  are  generally  associated  with  a  little 
mucus;  and,  in  men,  frequently  with  a  few  spermatozoa. 
Like  uric  acid,  their  chief  clinical  interest  lies  in  their 
tendency  to  form  calcuU,  and  their  presence  in  fresh 
urine,  together  with  evidences  of  renal  or  cystic  irritation, 
should  be  viewed  with  suspicion,  particularly  if  they  are 
clumped  in  small  masses. 

(4)  Leucin  and  T3n"0sin. — These  substances  are  cleav- 
age products  of  the  protein  molecule.  They  are  of  com- 
paratively rare  occurrence  in  the  urine  and  generally 
appear  together.  In  general,  their  presence  indicates 
autolysis  of  tissue  proteins.  Clinically,  they  are  seen 
most  frequently  in  severe  fatty  destruction  of  the  liver, 
such  as  occurs  in  acute  yellow  atrophy  and  phosphorus- 
poisoning.  Crystals  are  deposited  spontaneously  only 
when  the  substances  are  present  in  large  amount.  Usu- 
ally they  will  be  deposited  when  the  urine  is  evaporated 
to  a  small  volume  on  a  water-bath.  It  is  best,  however, 
to  separate  them  from  the  urine  as  follows: 

Treat  500  to  loooc.c.  of  urine,  which  has  been  freed  from 
albumin,  with  neutral,  then  with  basic,  lead  acetate  until  a 


MICROSCOPIC   EXAMINATION 


I«I 


precipitate  no  longer  forms.  Filter,  remove  excess  of  lead 
with  hydrogen  sulphid  (see  p.  170),  and  filter  again.  Con- 
centrate to  a  syrup  on  a  water-bath.  Extract  repeatedly 
with  small  quantities  of  absolute  alcohol  to  remove  urea. 
Treat  the  residue  with  hot  dilute  alcohol  to  which  a  little 
ammonia  has  been  added.  Filter  and  evaporate  the  filtrate 
to  a  small  volume  and  let  stand  for  the  leucin  and  tyrosin  to 
separate  out.  The  leucin  can  be  separated  from  the  tyrosin 
by  boiling  with  glacial  acetic  acid.  Leucin  dissolves,  leaving 
the  tyrosin,  and  can  again  be  recovered  by  evaporating  the 
acetic  acid. 

The  crystals  cannot  be  identified  from  their  morphol- 
ogy alone,  since  other  substances,  notably  calcium  phos- 
phate (see  Fig.  43)  and  ammonium  urate,  may  take 
similar  or  identical  forms.  It  is,  therefore,  necessary  to 
try  out  their  solubility  in  various  reagents  or  to  apply 
special  tests. 


Fig.  40. — Leucin  spheres  and  tyrosin  needles  (Stengel). 


Leucin  crystals  (Fig.  40)  as  they  appear  in  the  urine 
do  not  represent  the  pure  substance.  They  are  slightly 
yellow,  oily-looking  spheres,  many  of  them  with  radial 
and  concentric  striations.     Some  may  be  merged  to- 


l82  THE   URINE 

gether  in  clusters.    They  are  not  soluble  in  hydrochloric 
acid  nor  in  ether. 

Tyrosin  crystallizes  in  very  fine  needles,  which  may 
appear  black  and  which  are  usually  arranged  in  sheaves, 
with  a  marked  constriction  at  the  middle  (Fig.  40).  It 
is  soluble  in  ammonia  and  hydrochloric  acid,  but  not  in 
acetic  acid. 

Mbmer's  Test  for  Tyrosin. — To  a  small  quantity  of  the 
crystals  in  a  test-tube  add  a  few  cubic  centimeters  of 
Morner's  reagent  (formalin,  i  c.c;  distilled  water,  45  c.c; 
concentrated  sulphuric  acid,  55  c.c).  Heat  gently  to  the 
boiling-point.     A  green  color  shows  the  presence  of  tyrosin. 

(5)  Cystin  crystals  are  colorless,  highly  refractive, 
rather  thick,  hexagonal  plates  with  well-defined  edges. 
They  Ue  either  singly  or  superimposed  to  form  more  or 
less  irregular  clusters  (Fig.  41).  Uric  acid  sometimes 
takes  this  form  and  must  be  excluded.  Cystin  is  soluble 
in  hydrochloric  acid,  insoluble  in  acetic;  it  is  readily 
soluble  in  ammonia  and  recrystallizes  upon  addition  of 
acetic  acid. 

Cystin  is  one  of  the  amino-acids  formed  in  decompo- 
sition of  the  protein  molecule,  and  is  present  in  traces  in 
normal  urine.  Crystals  are  desposited  only  when  the  sub- 
stance is  present  in  excessive  amount.  Their  presence 
is  know-n  as  cystinuria.  It  is  a  rare  condition  due  to  an 
obscure  abnormality  of  protein  metabolism  and  usually 
continues  throughout  life.  There  are  rarely  any  symp- 
toms save  those  referable  to  renal  or  cystic  calculus,  to 
which  the  condition  strongly  predisposes. 

(6)  Fat-globules. — Fat  appears  in  the  urine  as  highly 
refractive  globules  of  various  sizes,  frequently  very  small. 


MICROSCOPIC   EXANINATION 


183 


These  globules  are  easily  recognized '  from  the  fact  that 
they  are  stained  black  by  osrtiic  acid  and  orange  or  red 
by  Sudan  III.  The  stain  may  be  appHed  upon  the  sUde, 
as  already  described  (see  p.  175).  Osmic  acid  should  be 
used  in  i  per  cent,  aqueous  solution;  Sudan  III,  in  satu- 
rated solution  in  70  per  cent,  alcohol,  to  which  one-half 
volume  of  10  per  cent,  formalin  may  advantageously 
be  added. 


\i.-^A*i 


Fig.  41. — Cystin  crystals  from  urine  of  patient  with  cystin  calculus  ( X  200)  (photograph 
by  the  author). 


Fat  in  the  urine  is  usually  a  contamination  from  un- 
clean vessels,  oiled  catheters,  etc.  A  very  small  amount 
may  be  present  after  ingestion  of  large  quantities  of  cod- 
liver  oil  or  other  fats.  In  fatty  degeneration  of  the 
kidney,  as  in  phosphorus-poisoning  and  chronic  paren- 
chymatous nephritis,  fat-globules  are  commonly  seen, 
both  free  in  the  urine  and  embedded  in  cells  and  tube- 


i84 


THE   URINE 


casts.     Fat-droplets  are  common  in  pus-corpuscles  and 
in  degenerating  cells  of  any  kind. 

In  chyluria,  or  admixture  of  chyle  with  the  urine  as  a 
result  of  rupture  of  a  lymph-vessel,  minute  droplets  of 
fat  are  so  numerous  as  to  give  the  urine  a  milky  appear- 
ance. The  droplets  are  smaller  than  those  of  milk,  which 
is  sometimes  added  by  malingerers.  The  fluid  is  often 
blood-tinged.  The  condition  is  best  recognized  by  shak- 
ing up  with  ether,  which,  when  separated,  leaves  the  urine 


Fig.  42. — Various  forms  of  triple  phosphate  crystals  (Ogden). 


comparatively  clear.  Chyluria  occurs  most  frequently 
as  a  symptom  of  infection  by  filaria  (see  p.  407),  the 
larvae  of  which  can  usually  be  found  in  the  milky 
urine. 

2.  In  Alkaline  Urine.— (i)  Phosphates.— While  most 
common  in  alkaline  urine,  phosphates  are  sometimes 
deposited  in  amphoteric  or  feebly  acid  urines.  The  usual 
forms  are:  (a)  Ammoniomagnesium  phosphate  crystals; 
(b)  acid  calcium  phosphate  crystals,  and  (c)  amorphous 
phosphates.    All  are  readily  soluble  in  acetic  acid. 


MICROSCOPIC   EXAMINATION 


185 


(a)  Ammoniomagnesium  Phosphate  Crystals. — They 
are  the  common  "triple  phosphate"  crystals,  which  are 
generally  easily  recognized  (Figs.  42  and  66,  and  Plate 
V).  They  are  colorless,  except  when  bile  stained. 
Their  usual  form  is  some  modification  of  the  prism,  with 
oblique  ends.  Most  typical  are  the  well-known  "coffin- 
lid"  and  "hip-roof"  forms.  The  long  axis  of  the  hip- 
roof crystal  is  often  so  shortened  that  it  resembles  the 
envelope  crystal  of  calcium  oxalate.  It  does  not,  how- 
ever, have  the  same  luster;  this,  and  its  solubility  in 
acetic  acid,  will  always  prevent  confusion. 


Fig.  43. — Crystals  of  calcium  phosphate:  i,  Common  form  (copied  from  Rieder's  Atlas); 
2,  needles  resembling  tyrosin  (drawn  from  nature);  3,  large,  irregular  plates  (from 
nature). 


When  rapidly  deposited,  as  by  artificial  precipitation, 
triple  phosphate  often  takes  feathery,  star-,  or  leaf-like 
forms.  These  gradually  develop  into  the  more  common 
prisms.  X-forms  may  be  produced  by  partial  solution  of 
prisms. 

{b)  Dicalcium  Phosphate  Crystals. — In  feebly  acid, 
.amphoteric,  or  feebly  alkaUne  urines  acid  calcium  phos- 
phate, wrongly  called  "neutral  calcium  phosphate," 
is  not  infrequently  deposited  in  the  form  of  colorless 


l86  THE   URINE 

prisms  arranged  in  stars  and  rosets  (Fig.  43,  i).  Because 
of  the  shape  of  the  crystals  it  is  sometimes  called  ''stellar 
phosphate."  The  individual  prisms  are  usually  slender, 
with  one  beveled,  wedge-like  end,  but  are  sometimes 
needle-like.  They  may  sometimes  take  forms  resembling 
ty rosin  (Fig.  43,  2),  calcium  sulphate,  or  hippuric  acid,  but 
are  readily  distinguished  by  their  solubility  in  acetic  acid. 
Calcium  phosphate  often  forms  large,  thin,  irregular, 
usually  granular,  colorless  plates,  which  are  easily  recog- 
nized (Fig.  43-  3)- 

a 

FJK.  44. — Indistinct  crystalline  sediment  (dumb-bell  crystals)  of  adcium  carbonate. 
Similar  crystals  are  sometimes  formed  by  calcium  oxalate  and  calcium  sulphate  (after 
I'unke). 

(c)  Amorphous  Phosphates. — The  earthy  phosphates 
are  thrown  out  of  solution  in  most  alkaline  and  many 
amphoteric  urines  as  a  white,  amorphous  sediment, 
which  may  be  mistaken  for  pus  macroscopically.  Under 
the  microscope  the  sediment  is  seen  to  consist  of  numer- 
ous colorless  granules,  distinguished  from  amorphous 
urates  by  their  color,  their  solubility  in  acetic  acid,  and 
the  reaction  of  the  urine. 

The  various  phosphatic  deposits  frequently  occur 
together.  They  are  sometimes  due  to  excessive  excre- 
tion of  phosphoric  acid,  but  usually  merely  indicate  that 
the  urine  has  become,  or  is  becoming,  alkaline.  (See 
Phosphates,  p.  109.) 


PLATE  V 


;>v 


Sediment    of   alkaline   fermentation    (after   Hofmann   and    Ultzmann). 


MICROSCOPIC   EXAMINATION  1 87 

(2)  Calcium  carbonate  may  sometimes  be  mingled 
with  the  phosphatic  deposits,  usually  as  amorphous 
granules,  or,  more  rarely,  as  colorless  spheres  and  dumb- 
bells (Fig.  44),  which  are  soluble  in  acetic  acid  with  gas 
formation. 

(3)  Ammonium  Urate  Crystals.- — This  is  the  only 
urate  deposited  in  alkaline  urine.  It  forms  opaque 
yellow  crystals,  usually  in  the  form  of  spheres  (Plate  V 
and  Fig.  66),  which  are  often  covered  with  fine  or  coarse 


Fig.  45. — Crystals  of  ammonium  urate  (one-half  of  the  forms  copied  from  Rieder's  Atlas; 
the  others,  from  nature). 

spicules — ' '  thorn-apple  crystals. ' '  Sometimes  dumb-bells, 
compact  sheaves  of  fine  needles,  and  irregular  rhizome 
forms  are  seen  (Fig.  45).  Upon  addition  of  acetic  acid 
they  dissolve,  and  rhombic  plates  of  uric  acid  appear. 

These  crystals  occur  only  when  free  ammonia  is 
present.  They  are  generally  found  along  with  the  phos- 
phates in  decomposing  urine  and  have  no  cHnical  sig- 
nificance. 

B.    Organized  Sediments 

The  principal  organized  structures  in  urinary  sedi- 
ments are:  Tube-casts;  epithehal  cells;  pus-corpuscles; 


1 88  THE   URINE 

red  blood-corpuscles;  spermatozoa;  bacteria,  and  animal 
parasites.  They  are  much  more  important  than  the 
unorganized  sediments  just  considered. 

1 .  Tube=casts. — These  interesting  structures  are  albu- 
minous casts  of  the  uriniferous  tubules.  Their  pres- 
ence in  the  urine  (known  as  cylindruria)  probably  always 
indicates  some  pathologic  change  in  the  kidney,  although 
this  change  may  be  very  slight  or  transitory.  Large 
numbers  may  be  present  in  temporary  irritations  and 
congestions.  They  do  not  in  themselves,  therefore,  imply 
organic  disease  of  the  kidney.  They  rarely  occur  in  urine 
which  does  not  contain,  or  has  not  recently  contained, 
albumin. 

While  it  is  not  possible  to  draw  a  sharp  dividing  line 
between  the  different  varieties,  casts  may  be  classified 
as  follows: 

(i)  Hyaline  casts.  . 
(a)  Narrow. 
(6)  Broad. 

(2)  Waxy  casts. 

(3)  Fibrinous  casts. 

(4)  Granular  casts. 

(a)  Finely  granular. 
{h)  Coarsely  granular. 

(5)  Fatty  casts. 

(6)  Casts  containing  organized  structures. 

(a)  Epithelial  casts. 
{h)  Blood-casts, 
(c)  Pus-casts. 
{d)  Bacterial  casts. 
As  will  be  seen  later,   practically   all  varieties  are 
modifications  of  the  hyaline. 


MICROSCOPIC   EXAMINATION  1 89 

The  significance  of  the  different  varieties  is  more 
readily  understood  if  one  considers  their  mode  of  forma- 
tion. Albuminous  material,  the  source  and  nature  of 
which  are  not  definitely  known,  but  which  are  doubtless 
not  the  same  in  all  cases,  probably  enters  the  lumen  of  a 
uriniferous  tubule  in  a  fluid  or  plastic  state.  The  ma- 
terial has  been  variously  thought  to  be  an  exudate 
from  the  blood,  a  pathologic  secretion  of  the  renal  cells, 
and  a  product  of  epithelial  degeneration.  In  the  tubule 
it  hardens  into  a  cast  which,  when  washed  out  by  the 
urine,  retains  the  shape  of  the  tubule,  and  contains 
within  its  substance  whatever  structures  and  debris  were 
lying  free  within  the  tubule  or  were  loosely  attached  to 
its  wall.  If  the  tubule  be  small  and  has  its  usual  hning 
of  epithelium,  the  cast  will  be  narrow;  if  it  be  large  or 
entirely  denuded  of  epitheHum,  the  cast  will  be  broad. 
A  cast,  therefore,  indicates  the  condition  of  the  tubule  in 
which  it  is  formed,  but  does  not  necessarily  indicate  the 
condition  of  the  kidney  as  a  whole. 

The  search  for  casts  must  be  carefully  made.  The 
urine  must  be  fresh,  since  hyaline  casts  soon  dissolve 
when  it  becomes  alkaline.  It  should  be  thoroughly 
centrifugalized.  When  the  sediment  is  abundant,  casts, 
being  light  structures,  will  be  found  near  the  top.  In 
cystitis,  where  casts  may  be  entirely  hidden  by  the  pus, 
the  bladder  should  be  irrigated  to  remove  as  much  of 
the  pus  as  possible  and  the  next  urine  examined.  In 
order  to  prevent  solution  of  the  casts  the  urine,  if  alka- 
line, must  be  rendered  acid  by  previous  administra- 
tion of  boric  acid  or  other  drugs.  Heavy  sediments  of 
urates,  blood,  or  vaginal  cells  may  likewise  obscure 
casts  and  other  important  structures.    The  last  can  be 


190  THE    URINE 

avoided  by  catheterization.  Urates  can  be  dissolved 
by  gently  warming  before  centrifugalizing,  care  being 
taken  not  to  heat  enough  to  coagulate  the  albumin. 
The  aluminum  shield  of  the  centrifuge  tube  may  also 
be  heated.  Blood  can  be  destroyed  by  centrifugalizing, 
pouring  off  the  supernatant  urine,  filling  the  tube  with 
water,  adding  a  few  drops  of  dilute  acetic  acid,  mixing 
well,  and  again  centrifugalizing;  this  process  being  re- 
peated until  the  blood  is  completely  decolorized.  Too 
much  acetic  acid  will  dissolve  hyaline  casts. 

Their  cylindric  shape  can  be  best  seen  by  slightly 
moving  the  cover-glass  while  observing  them,  thus  caus- 
ing them  to  roll.  This  little  manipulation  should  be 
practised  until  it  can  be  done  satisfactorily.  It  will 
l^rove  useful  in  many  examinations. 

Various  methods  of  staining  casts  so  as  to  render  them 
more  conspicuous  have  been  proposed.  They  offer  no 
special  advantage  to  one  who  understands  how  to  use 
the  substage  mechanism  of  his  microscope.  The  "nega- 
tive-staining" method  is  as  good  as  any.  It  consists 
simply  in  adding  a  little  India-ink  to  the  drop  of  urine 
on  the  slide.  Casts,  cells,  etc.,  will  stand  out  as  colorless 
structures  on  a  dark  background. 

(i)  Hyaline  Casts. — Typically,  these  are  colorless, 
homogeneous,  semitransparant,  cylindric  structures,  with 
parallel  sides  and  usually  rounded  ends.  Not  infre- 
quently they  are  more  opaque  or  show  a  few  granules  or 
an  occasional  oil-globule  or  cell,  either  adhering  to  them 
or  contained  within  their  substance.  Generally  they 
are  straight  or  curved;  less  commonly,  convoluted. 
Their  length  and  breadth  vary  greatly:  they  are  some- 
times so  long  as  to  extend  across  several  fields  of  a 


MICROSCOPIC   EXAMINATION 


191 


medium-power  objective,  but  are  usually  much  shorter; 
in  breadth   they  vary  from  one  to  seven  or  eight  times 


Fig.  46. — Hyaline  casts  showing  fat-droplets  and  leukocytes  (obj.  4  mm.)  (Boston). 

the  diameter  of  a  red  blood-corpuscle.    (See  Figs.  4,  46, 
47,  and  51.) 


Fig.  47.— Various  kinds  of  casts:  a,  Hyaline  and  finely  granular  cast;  6,  finely  granular 
cast;  f,  coarsely  granular  cast;  d,  brown  granular  cast;  e,  granular  cast  with  normal  and 
abnormal  blood  adherent;/,  granular  cast  with  renal  cells  adherent;  g,  granular  cast  with 
fat  and  a  fatty  renal  cell  adherent  (Ogden). 


Hyaline  casts  are  the  least  significant  of  all  the  casts, 
and  occur  in  many  slight  and   transitory   conditions. 


192  THE   URINE 

Small  numbers  are  common  following  ether  anesthesia, 
in  fevers,  after  excessive  exercise,  and  in  congestions  and 
irritations  of  the  kidney.  They  are  always  present,  and 
are  usually  stained  yellow  when  the  urine  contains  much 
bile.  While  they  are  found  in  all  organic  diseases  of  the 
kidney,  they  are  most  important  in  chronic  interstitial 
nephritis.  Here  they  are  seldom  abundant,  but  their 
constant  presence  is  the  most  reliable  urinary  sign  of  the 
disease.  Small  areas  of  chronic  interstitial  change  are 
probably  responsible  for  the  few  hyaline  casts  so  fre- 
quently found  in  the  urine  of  elderly  persons. 


Fig.  48. — Waxy  casts  (upper  part  of  figure).    Fatty  and  fat-bearing  casts  (lower  part  of 
figure)  (from  Greene's  "Medical  Diagnosis"). 

Very  broad  hyaline  casts  commonly  indicate  complete 
desquamation  of  the  tubular  epithelium,  such  as  occurs 
in  the  late  stages  of  nephritis. 

(2)  Waxy  Casts. — Like  hyaline  casts,  these  are  homo- 
geneous when  typical,  but  frequently  contain  a  few 
granules  or  an  occasional  cell.  They  are  much  more 
opaque  than  the  hyaline  variety,  and  are  usually  shorter 
and  broader,  with  irregular,  broken  ends,  and  some- 
times appear  to  be  segmented.  They  are  grayish  or 
colorless,  and  have  a  dull,  waxy  look,  as  if  cut  from  par- 


MICROSCOPIC   EXAMINATION  1 93 

aflSn  (Figs.  48  and  64).  They  are  sometimes  composed 
of  material  which  gives  the  amyloid  reactions.  All 
gradations  between  hyaline  and  waxy  casts  may  be  found. 
Waxy  casts  are  found  in  most  advanced  cases  of  nephritis, 
where  they  are  an  imfavorable  sign.  They  are  perhaps 
most  abundant  in  amyloid  disease  of  the  kidney,  but 
are  not  distinctive  of  the  disease,  as  is  sometimes  stated. 

(3)  Fibrinous  Casts. — Casts  which  resemble  waxy 
casts,  but  have  a  distinctly  yellow  color,  as  if  cut  from 
beeswax,  are  often  seen  in  acute  nephritis.  They  are 
called  fibrinous  casts,  but  the  name  is  inappropriate,  as 
they  are  not  composed  of  fibrin.  They  are  often  classed 
with  waxy  casts,  but  should  be  distinguished,  as  their 
significance  is  much  less  serious. 

(4)  Granular  Casts. — These  are  merely  hyaline  casts 
in  which  numerous  granules  are  embedded  (Figs.  47,  49, 
and  51). 

Finely  granular  casts  contain  many  fine  granules,  are 
usually  shorter,  broader,  and  more  opaque  than  the 
hyaline  variety,  and  are  more  conspicuous.  Their  color 
is  grayish  or  pale  yellow. 

Coarsely  granular  casts  contain  larger  granules  and  are 
darker  in  color  than  the  finely  granular,  being  often 
dark  brown,  owing  to  presence  of  altered  blood-pigment. 
They  are  usually  shorter  and  more  irregular  in  outhne, 
and  more  frequently  have  irregularly  broken  ends. 

(5)  Fatty  Casts. — Small  droplets  of  fat  may  at  times 
be  seen  in  any  variety  of  cast.  Those  in  which  the  drop- 
lets are  numerous  are  called  fatty  casts  (Figs.  48  and  49) . 
The  fat-globules  are  not  difficult  to  recognize.  Staining 
with  osmic  acid  or  Sudan  (see  p.  183)  will  remove  any 
doubt  as  to  their  nature. 

13 


194 


THE   URINE 


The  granules  and  fat-droplets  seen  in  casts  are  prod- 
ucts of  epithelial  degeneration.  Granular  and  fatty 
casts,  therefore,  always  indicate  partial  or  complete  dis- 
integration of  the  renal  epithelium.  The  finely  granular 
variety  is  the  least  significant,  and  is  found  when  the 
epithelium  is  only  moderately  affected.  Coarsely 
granular,  and  especially  fatty  casts,  if  present  in  con- 
siderable numbers,  indicate  a  serious  parenchymatous 
nephritis. 

(6)  Casts  Containing  Organized  Structures. — Cells 
and  other  structures  are  frequently  seen  adherent  to  a 


Fig.  40. — Granular  and  fatty  casts  and  two  compound  granule  cells  (Stengel). 


cast  or  embedded  within  it.     (See  Figs.  46  and  47.) 
When  numerous,  they  give  name  to  the  cast. 

(a)  Epithelial  casts  contain  epithelial  cells  from  the 
renal  tubules.  The  cells  vary  in  size  and  are  often 
flattened,  oval,  or  elongated.  They  may  be  recognized 
as  epithelial  cells  by  irrigating  with  dilute  acetic  acid, 
which  brings  out  the  nucleus  clearly.  Epithelial  casts 
always  imply  desquamation  of  epithelium,  which  rarely 
occurs   except   in    parenchymatous   inflammations  (see 


MICROSCOPIC   EXAMINATION 


195 


Figs.  63  and  64).     When  the  cells  are  well  preserved 
they  point  to  acute  nephritis. 

(b)  Blood-casts  contain  red  blood-corpuscles,  usually 
much  degenerated  (Figs.  50  and  63).  They  always 
indicate  hemorrhage  into  the  tubules,  which  is  most 
common  in  acute  nephritis  or  an  acute  exacerbation  of 
a  chronic  nephritis. 

(c)  Pus-casts  (see  Fig.  65),  composed  almost  wholly 
of  pus-corpuscles,  are  uncommon,  and  point  to  a  chronic 
suppurative  process  in  the  kidney. 


Fig.  50. — Red  blood-corpuscles  and  blood-casts  (courtesy  of  Dr.  A.  Scott)  (obj.  4  mm.) 

(Boston). 

(d)  True  bacterial  casts  are  rare.  They  indicate  a 
septic  condition  in  the  kidney.  Bacteria  may  permeate 
a  cast  after  the  urine  is  voided. 

Structures  Likely  to  Be  Mistaken  for  Casts. — (i) 
Mucous  Threads. — Mucus  frequently  appears  in  the 
.form  of  long  strands  which  slightly  resemble  hyaline 
casts  (Fig.  51).  They  are,  however,  more  ribbon-like, 
have   less  well-defined   edges,   and   usually   show   faint 


196 


THE  URINE 


longitudinal  striations.  Their  ends  taper  to  a  point  or 
are  split  or  curled  upon  themselves,  and  are  never  evenly 
rounded,  as  is  commonly  the  case  with  hyaline  casts. 

Such  threads  form  a  part  of  the  nubecula  of  normal 
urine,  and  are  especially  abundant  when  calcium  oxalate 
crystals  are  present.  When  there  is  an  excess  of  mucus, 
as  in  irritations  of  the  urinary  tract,  every  field  may  be 
filled  with  an  interlacing  meshwork. 


■vX. 


■iSS^- 


d^ 


IL 


*/- 


Fig.  51. — Hyaline  and  granular  casts,  mucous  threads,  and  cylindroids.    There  are  also 
a  few  epithelial  cells  from  the  bladder  (Wood). 


Mucous  threads  are  microscopic  and  should  not  be 
confused  with  urethral  shreds  or  "gonorrheal  threads," 
which  are  macroscopic,  0.5  to  i  cm.  long,  and  consist  of  a 
matrix  of  mucus  in  which  many  epithelial  and  pus-cells 
are  embedded. 

(2)  Cylindroids. — This  name  is  sometimes  given  to  the 
mucous  threads  just  described,  but  is  more  properly 
applied  to  certain  peculiar  structures  more  nearly  allied 
to  casts.    They  resemble  hyaline  casts  in  structure,  but 


MICROSCOPIC  EXAMINATION  1 97 

differ  in  being  broader  at  one  end  and  tapering  to  a 
slender  tail,  which  is  often  twisted  or  curled  upon  itself 
(Fig.  51).  They  frequently  occur  in  the  urine  along 
with  hyaline  casts,  especially  in  irritations  of  the  kidney, 
and  have  practically  the  same  significance. 

(3)  Masses  of  amorphous  urates,  or  phosphates,  or 
very  small  crystals  (Fig.  52),  which  accidentally  take  a 
cyHndric  form,  or  shreds  of  mucus  covered  with  granules, 
closely  resemble  granular  casts.     Application  of  gentle 


Fig.  S2- — Calcium  oxalate  crystals,  showing  a  pseudocast  of  small  crystals  (Jakob). 

heat  or  appropriate  chemicals  will  serve  to  differentiate 
them.  When  urine  contains  both  mucus  and  granules, 
large  numbers  of  these  "pseudocasts,"  all  lying  in  the 
same  direction,  can  be  produced  by  slightly  moving  the 
cover-glass  from  side  to  side.  It  is  possible — as  in  urate 
infarcts  of  infants — for  urates  to  be  molded  into  cyUn- 
-dric  bodies  within  the  renal  tubules. 

(4)  Hairs   and  fibers   of  wool,   cotton,   etc.     These 
could  be  mistaken  for  casts  only  by  beginners.     One 


198  THE   URINE 

can  easily  become  familiar  with  their  appearance  by 
suspending  them  in  water  and  examining  with  the  micro- 
scope (see  Fig.  61). 

(5)  Hyphae  of  molds  are  not  infrequently  mistaken 
for  hyaline  casts.  Their  higher  degree  of  refraction, 
their  jointed  or  branching  structure,  and  the  accom- 
panying spores  will  differentiate  them  (see  Fig.  62). 

2.  Epithelial  Cells. — A  few  cells  from  various  parts 
of  the  urinary  tract  occur  in  every  urine.  A  marked 
increase  indicates  some  pathologic  condition  at  the  site 
of  their  origin.  It  is  sometimes,  but  by  no  means  always, 
possible  to  locate  their  source  from  their  form.  One 
should,  however,  be  extremely  cautious  about  making 
any  definite  statement  as  to  the  origin  of  any  individual 
cell.  Most  cells  are  much  altered  from  their  original 
shape.  Any  epithelial  cell  may  be  so  granular  from 
degenerative  changes  that  the  nucleus  is  obscured. 
Most  of  them  contain  fat-globules.  They  are  usually 
divided  into  three  groups : 

(i)  Small,  round  or  polyhedral  cells  are  about  the 
size  of  pus-corpuscles,  or  a  Httle  larger,  with  a  single 
round  nucleus.  Such  cells  may  come  from  the  deeper 
layers  of  any  part  of  the  urinary  tract.  They  are  uncom- 
mon in  normal  urine.  When  they  are  dark  in  color,  very 
granular,  and  contain  a  comparatively  large  nucleus, 
they  probably  come  from  the  renal  tubules,  but  their 
origin  in  the  kidney  is  not  proved  unless  they  are  found 
embedded  in  casts.  In  chronic  passive  congestion  of 
the  kidney  some  of  these  cells  may  contain  yellow 
granules  of  altered  blood-pigment.  They  are  analogous 
to  the  "heart-failure  cells"  of  the  sputum  (see  p.  79). 
Renal  cells  are  abundant  in  parenchymatous  nephritis. 


MICROSCOPIC   EXAMINATION 


199 


^ 


especially  the  acute  form.  They  are  nearly  always  fatty 
— ^most  markedly  so  in  chronic  parenchymatous  nephri- 
tis, where  their  substance  is  sometimes  wholly  replaced 
by  fat-droplets  ("compound  gran- 
ule cells")   (see  Figs.  49,  53,  and 

63). 

(2)  Irregular  cells  are  consider- 
ably larger  than  the  preceding. 
They  are  round,  pear  shaped,  or 
spindle  shaped,  or  may  have  tail- 
like processes,  and  are  hence 
named  large  round,  pyriform, 
spindle,  or  caudate  cells  respect- 
iveLy.  Each  contains  a  round 
or  oval  distinct  nucleus.  Their 
usual  source  is  the  deeper  layers 
of  the  urinary  tract,  especially 
of  the  bladder.  Caudate  forms 
come  most  commonly  from  the  pelvis  of  the  kidney  (see 
Figs.  54,  ^,  55'  65,  and  66). 

(3)  Squamous  or  pavement  cells  are  large  flat  cells, 
each  with  a  small,  distinct  round  or  oval  nucleus  (Fig. 
54,  a).  They  are  derived  from  the  superficial  layers  of 
the  ureters,  bladder,  urethra,  or  vagina,  and  when 
desquamation  is  active,  appear  in  stratified  masses. 
Squamous  cells  from  the  bladder  are  generally  rounded, 
while  those  from  the  vagina  are  larger,  thinner,  and 
more  angular.  Great  numbers  of  these  vaginal  cells, 
together  with  pus-corpuscles,  may  be  present  when 
leukorrhea  exists. 

3.  Pus=corpuscIes. — A  very  few  leukocytes  are  pres- 
ent in  normal  urine.     They  are  more  abundant  when 


F'S-  53- — Renal  epithelium 
from  nephritic  urine:  a,  Poly- 
hedral epithelium  in  nephritis 
of  scariet  fever;  b  and  c,  differ- 
ent grades  of  fatty  degenera- 
tion in  renal  epithelium  in 
chronic  nephritis  (X  400) 
(after  Bizzozero). 


200  THE   URINE 

mucus  is  present.  An  excess  of  leukocytes,  mainly  of 
the  polymorphonuclear  neutrophilic  variety,  with  albu- 
min, constitutes  pyuria — pus  in  the  urine. 


Fig-  54- — Epithelial  cells  from  urethra  and  bladder:  a,  Squamous  cells  from  superficial 
layers;  b,  irregular  cells  from  deeper  layers  Qakob). 


Fig.  55. — Caudate  epithelial  cells  from  pelvis  of  kidney  (Jakob). 

When  at  all  abundant,  pus  forms  a  white  sediment 
resembling  amorphous  phosphates  macroscopically.    Un- 


MICROSCOPIC  EXAMINATTON  20I 

der  the  microscope  the  corpuscles  appear  as  very  granu- 
lar cells,  about  twice  the  diameter  of  a  red  blood-cor- 
puscle (Figs.  56  and  66).  The  granules  are  partly  the 
normal  neutrophilic  granules,  partly  granular  products 
of  degeneration.  In  freshly  voided  urine  many  exhibit 
ameboid  motion,  assuming  irregular  outlines.  Each 
contains  one  irregular  nucleus  or  several  small,  rounded 
nuclei.  The  nuclei  are  obscured  or  entirely  hidden  by 
the  granules,  but  may  be  brought  clearly  into  view  by 
running  a  little  acetic  acid  under  the  cover-glass.  This 
enables  one  to  easily  distinguish  pus-corpuscles  from 


i 


®    @    ® 


© 


M  ^^  ^    ®    ©  ® 


Fig.  56. — Pus-corpuscles:  a,  As  ordinarily  seen;  6,  ameboid  corpuscles;  c,  showing  the 
action  of  acetic  acid  (Ogden). 

small  round  epithelial  cells,  which  resemble  them  in  size, 
but  have  a  single,  rather  large,  round  nucleus.  In  de- 
composing urine  pus  is  often  converted  into  a  gelatinous 
mass  which  gives  the  urine  a  ropy  consistence. 

Pyuria  indicates  suppuration  in  some  part  of  the 
urinary  tract — urethritis,  cystitis,  pyelitis,  etc. — or  may 
be  due  to  contamination  from  the  vagina,  in  which  case 
many  vaginal  epithelial  cells  will  also  be  present.  In 
general,  the  source  of  the  pus  can  be  determined  only  by 
the  accompanying  structures  (epithelia,  casts)  or  by  the 

clinical  signs. 

H  L  ix 

COLLlKilE  01"   vo  .L  ^'i  /. 


202  THE    URINE 

A  fairly  accurate  idea  of  the  quantity  of  pus  from  day 
to  day  may  be  had  by  shaking  the  urine  thoroughly  and 
counting  the  number  of  corpuscles  per  cubic  milhmeter 
upon  the.  blood-counting  slide.  A  drop  of  the  urine 
is  placed  directly  upon  the  slide.  Dilution  is  seldom 
necessary.  The  urine  must  not  be  alkaline  or  the  cor- 
puscles will  adhere  in  clumps. 

Pus  always  adds  a  certain  amount  of  albumin  to  the 
urine,  and  it  is  often  desirable  to  know  whether  the 
albumin  present  in  a  given  specimen  is  due  solely  to 
pus.  It  has  been  estimated  that  80,000  to  100,000  pus- 
corpuscles  per  cubic  millimeter  add  about  o.i  per  cent. 
of  albumin.  If  albumin  is  present  in  much  greater  pro- 
portion than  this,  the  excess  is  probably  derived  from  the 
kidney. 

4.  Red  Blood=corpuscles. — Urine  which  contains  blood 
is  always  albuminous.  Very  small  amounts  do  not  alter 
its  macroscopic  appearance.  Larger  amounts  alter  it 
considerably.  Blood  from  the  kidneys  is  generally  inti- 
mately mixed  with  the  urine  and  gives  it  a  hazy  reddish 
or  brown,  ''smoky"  color.  When  from  the  lower  urinary 
tract,  it  is  not  so  intimately  mixed  and  settles  more 
quickly  to  the  bottom,  the  color  is  brighter,  and  small 
clots  are  often  present. 

Red  blood-corpuscles  are  not  usually  difficult  to  recog- 
nize with  the  microscope.  When  very  fresh,  they  have  a 
normal  appearance,  being  yellowish  disks  of  uniform 
size  (normal  blood).  When  they  have  been  in  the  urine 
any  considerable  time,  their  hemoglobin  may  be  dis- 
solved out,  and  they  then  appear  as  faint  colorless  circles 
or  "shadow  cells"  (abnormal  blood),  and  are  more 
difficult  to  see  (Fig.  57;  see  also  Figs.  50  and  63).    They 


MICROSCOPIC  EXAMINATION  203 

are  apt  to  be  swollen  in  dilute  and  crenated  in  concen- 
trated urines.  The  microscopic  findings  may  be  cor- 
roborated by  chemic  tests  for  hemoglobin,  although  the 
microscope  may  show  a  few  red  corpuscles  when  the 
chemic  tests  are  negative. 

When  not  due  to  contamination  from  menstrual  dis- 
charge, blood  in  the  urine,  or  hematuria,  is  always  patho- 
logic. Blood  comes  from  the  kidney  tubules  in  severe 
hyperemia,  in  acute  nephritis  and  acute  exacerbations  of 
chronic  nephritis,  and  in  renal  tuberculosis  and  malig- 
nant disease.  An  "idiopathic  hematuria,"  probably  of 
nervous  origin,  has  been  observed.    The  finding  of  blood- 

^        O  ^    '^    o  "rT      ^   ° 
Of.         ^^  ^000. 

*        O^      ^       o  O      ^ 

Fig-  57- — Blood-corpuscles:  a,  Normal;  6,  abnormal  (Ogden). 

casts  is  the  only  certain  means  of  diagnosing  the  kidney 
as  its  source.  Blood  comes  from  the  pelvis  of  the  kidney 
in  renal  calculus  (see  Fig.  65),  and  is  then  usually  inter- 
mittent, small  in  amount,  and  accompanied  by  a  Kttle  pus 
and  perhaps  crystals  of  the  substance  forming  the  stone. 
Considerable  hemorrhages  from  the  bladder  may  occur 
in  vesical  calculus,  tuberculosis,  and  new  growths. 
Small  amounts  of  blood  generally  accompany  acute 
cystitis.  In  Africa  the  presence  of  Schistosomum  hcema- 
tobium  in  the  veins  of  the  bladder  is  a  common  cause  of 
.hemorrhage  (Egj^tian  hematuria). 

It  is  sometimes  possible  to  gain  a  clue  to  the  site  of 
the  bleeding  by  having  the  patient  pass  his  urine  in 


204  THE  T7RINE 

three  separate  portions.  If  the  blood  be  chiefly  in  the 
first  portion,  the  bleeding  point  is  probably  in  the  ure- 
thra; if  in  the  last,  it  is  probably  in  the  bladder.  If  the 
blood  is  uniformly  mixed  in  all  three  portions,  it  prob- 
ably comes  from  the  kidney  or  ureter. 

5.  Spermatozoa  are  generally  present  in  the  urine  of 
men  after  nocturnal  emissions,  after  epileptic  convul- 
sions, and  in  spermatorrhea.    They  may  be  found  in  the 


? 


w  — r  \ 


\ 


C  ^.  ^ 


^ 


Fig.  s8. — Spermatozoa  in  urine  (Ogden). 

urine  of  both  sexes  following  coitus.  They  are  easily 
recognized  from  their  characteristic  structure  (Fig.  58). 
The  4-mm.  objective  should  be  used,  with  subdued  light 
and  careful  focusing. 

6.  Bacteria. — Normal  urine  is  free  from  bacteria  in 
the  bladder,  but  becomes  contaminated  in  passing 
through  the  urethra.  Various  non-pathogenic  bacteria 
are  always  present  in  decomposing  urine.    In  suppura- 


MICROSCOPIC  EXAMINATION  205 

tions  of  the  urinary  tract  pus-producing  organisms  may- 
be found.  In  many  infectious  diseases  the  specific 
bacteria  may  be  ehminated  in  the  urine  without  pro- 
ducing any  local  lesion.  Typhoid  bacilli  have  been 
known  to  persist  for  months  and  even  years  after  the 
attack. 

Bacteria  produce  a  cloudiness  which  will  not  clear 
upon  filtration.  They  are  easily  seen  with  the  4-mm. 
objective  in  the  routine  microscopic  examination. 
Ordinarily,  no  attempt  is  made  to  identify  any  but  the 
tubercle  bacillus  and  the  gonococcus. 

Tubercle  bacilli  are  nearly  always  present  in  the  urine 
when  tuberculosis  exists  in  any  part  of  the  urinary  tract, 
but  are  often  difficult  to  find,  especially  when  the  urine 
contains  little  or  no  pus. 

Detection  of  Tubercle  Bacilli  in  Urine. — ^The  urine  should 
be  obtained  by  catheter  after  careful  cleansing  of  the  parts. 

(i)  Centrifugalize  thoroughly,  after  dissolving  any  sedi- 
ment of  urates  or  phosphates  by  gentle  heat  or  acetic  acid. 
Pour  off  the  supernatant  fluid,  add  water,  and  centrifugalize 
again.  Addition  of  i  or  2  volumes  of  alcohol  will  favor 
centrifugalization  by  lowering  the  specific  gravity. 

(2)  Make  thin  smears  of  the  sediment,  adding  a  little  egg- 
albumen  if  necessary  to  make  the  smear  adhere  to  the  glass; 
dry,  and  fix  in  the  usual  way. 

(3)  Stain  with  carbolfuchsin,  steaming  for  at  least  three 
minutes,  or  at  room  temperature  for  six  to  twelve  hours. 

(4)  Wash  in  water,  and  then  in  20  per  cent,  nitric  acid, 
until  only  a  faint  pink  color  remains. 

-    (5)  Wash  in  water. 

(6)  Soak  in  alcohol  fifteen  minutes  or  longer.  This  decolor- 
izes the  smegma  bacillus  (see  p.  69),  which  is  often  present  in 


206  THE   URINE 

the  urine,  and  might  easily  be  mistaken  for  the  tubercle  bacil- 
lus. It  is  unlikely,  however,  to  be  present  in  catheterized 
specimens.  It  is  always  safest  to  soak  the  smear  in  alcohol 
for  several  hours  or  over  night,  since  some  strains  of  the 
smegma  bacillus  are  very  resistant. 

(7)  Wash  in  water. 

(8)  Apply  Loffler's  methylene  blue  solution  for  one-half 
minute. 

(9)  Rinse  in  water,  dry  between  filter-papers,  and  examine 
with  the  oil-emersion  objective. 

When  the  bacilli  are  scarce,  the  following  method  may  be 
tried.  It  is  applicable  also  to  other  fluids.  If  the  fluid  is  not 
albuminous,  add  a  little  egg-albumen.  Coagulate  the  albu- 
men by  gentle  heat  and  centrifugalize.  The  bacilli  will  be 
carried  down  with  the  albumen.  The  sediment  is  then  treated 
by  the  antiformin  method  (see  p.  68). 

A  careful  search  of  many  smears  may  be  necessarv-  to  find 
.the  bacilli.  They  usually  lie  in  clusters  (see  Plate  VI).  Fail- 
ure to  find  them  in  suspicious  cases  should  be  followed  by 
inoculation  of  guinea-pigs;  this  is  the  court  of  last  appeal, 
and  must  also  be  sometimes  resorted  to  in  order  to  exclude 
the  smegma  bacillus. 

In  gonorrhea,  gonococci  are  sometimes  found  within 
pus-cells  in  the  sediment,  but  more  commonly  in  the 
"gonorrheal  threads"  or  "floaters."  In  themselves, 
these  threads  are  by  no  means  diagnostic  of  gonorrhea. 
They  are  most  common  in  the  morning  or  after  massage 
of  the  prostate.  Detection  of  the  gonococcus  is  de- 
scribed later  (see  p.  422). 

7.  Animal  parasites  are  rare  in  the  urine.  Hooklets 
and  scoHces  of  Tania  echinococcus  (Fig.  59)  and  larvae 
of  filariae  have  been  met.  •  In  Africa  the  ova,  and 
even  adults,  of  Schistosomum  hcematobium  are  common, 


PLATE  VI 


J  If 


0^      ■■% 


»»^ 


^ 


*fe 


Tubercle  bacilli  in  urinary  sediment;  X  Soo  (Ogden). 


MICKOSCOPIC   EXAMINATION 


207 


accompanying    "Egyptian    hematuria."      Trichomonas 
vaginalis  is  a  not  uncommon  contamination.    This  and 


Fig-  59- — 1>  Scolex  of  Tsenia  echinococcus,  showing  crown  of  booklets;  2,  scolex  and  de- 
tached booklets  (obj.  4  mm.)  (Boston). 

other  protozoa  may  be  mistaken  for  spermatozoa  by  the 
inexperienced. 


Fig.  60. — Embryo  of  "vinegar  eel"  in  urine,  from  contamination;  length,  340  fi;  width, 
IS  /«..  An  epithebal  cell  from  bladder  and  three  leukocytes  are  also  shown  (studied  with 
Dr.  J.  A.  Wilder). 

A  worm  which  is  especially  interesting  is  Anguillula 
aceti,  the  "vinegar  eel."     This  is  generally  present  in 


2o8  THE  URINE 

the  sediment  of  table  vinegar,  and  may  reach  the  urine 
through  use  of  vinegar  in  vaginal  douches,  or  through 
contamination  of  the  bottle  in  which  the  urine  is  con- 
tained. It  has  been  mistaken  for  Strongyloides  intes- 
tinalis  and  for  the  larval  filaria.  It  closely  resembles 
the  former  in  both  adult  and  embryo  stages.  The  young 
embryos  have  about  the  same  length  as  the  larvae  of 
Filaria  bancrofti,  but  are  nearly  twice  as  broad,  and  the 
intestinal  canal  is  easily  seen  (compare  Figs.  60  and  134). 
For  fuller  descriptions  of  these  parasites  the  reader 
is  referred  to  Chapter  VI. 

C   Extraneous  Structures 

The  laboratory  worker  must  familiarize  himself  with 
the  microscopic  appearance  of  the  more  common  of  the 
numerous  structures  which  may  be  present  from  acci- 
dental contamination  (Fig.  61). 

Yeast-cells  are  smooth,  colorless,  highly  refractive, 
spheric  or  ovoid  cells.  They  sometimes  reach  the  size  of 
a  leukocyte,  but  are  generally  smaller  (see  Fig.  106,  /). 
They  might  be  mistaken  by  the  inexperienced  for  red 
blood-corpuscles,  fat-droplets,  or  the  spheric  crystals  of 
calcium  oxalate,  but  are  distinguished  by  the  facts  that 
they  are  not  of  uniform  size;  that  they  tend  to  adhere  in 
short  chains;  that  small  buds  may  often  be  seen  ad- 
hering to  the  larger  cells;  and  that  they  do  not  give  the 
hemoglobin  test,  are  not  stained  by  osmic  acid  or  Sudan, 
but  are  colored  brown  by  Lugol's  solution,  and  are  in- 
soluble in  acids  and  alkalis.  Yeast-cells  multiply  rapidly 
in  diabetic  urine,  and  may  reach  the  bladder  and  multi- 
ply there. 


MICROSCOPIC   EXAMINATION 


209 


Mold  fungi  (Fig.  62)  are  characterized  by  refractive, 
jointed,  or  branched  rods  (hyphae),  often  arranged  in  a 
network,  and  by  highly  refractive  spheric  or  ovoid 
spores.  They  are  common  in  urine  which  has  stood 
exposed  to  the  air. 


Fig.  61. — Extraneous  matters  found  in  urine:  a,  Flax-fibers;  6,  cotton-fibers;  c,  feathers; 
d,  hairs;  e,  pwtato-starch;/,  rice-starch  granules;  g.wheat-starch;  A,  air-bubbles;  t,  muscular 
tissue;  k,  vegetable  tissue;  /,  oil-globules. 

Fibers  of  wool,  cotton,  Hnen,  or  silk,  derived  from 
towels,  the  clothing  of  the  patient,  or  the  dust  in  the  air, 
are  present  in  almost  every  urine.  Fat-droplets  are 
most  frequently  derived  from  unclean  bottles  or  oiled 
catheters.  Starch-granules  may  reach  the  urine  from 
towels,  the  clothing,  or  dusting-powders.  They  are 
recognized  by  their  concentric  striations  and  their  blue 

14 


2IO  THE   URINE 

color  with  iodin  solution.  Lycopodium  granules  (see 
Fig.  5)  may  also  reach  the  urine  from  dusting-powders. 
They  might  be  mistaken  for  the  ova  of  parasites.  Bub- 
bles of  air  (see  Fig.  61,  h)  are  often  confusing  to  be- 
ginners, but  are  easily  recognized  after  once  being  seen. 
Scratches  and  flaws  in  the  glass  of  slide  or  cover  are 
often  most  assiduously  studied  by  beginners,  and  are  not 
infrequently  reported  as  rare  crystals,  tube-casts,  or  even 
worms.  Dirt  upon  the  cover  (especially  when  this  is  taken 
directly  from  the  original  box  without  cleaning)  is  like- 


Fig.  62. — Aspergillus  from  urine  (Boston). 

wise  a  common  source  of  confusion.  Fibers  of  muscle 
(Figs.  61,  i,  and  iii)  and  other  particles  which  are  evi- 
dently of  fecal  origin  are  usually  the  result  of  contamina- 
tion, but  may  rarely  be  present  in  catheterized  specimens. 
They  then  indicate  rectovesical  fistula. 

IV.  THE  URINE  IN  DISEASE 

In  this  section  the  characteristics  of  the  urine  in  those 
diseases  which  produce  distinctive  urinary  changes  will 
be  briefly  reviewed. 


THE   URINE   IN   DISEASE  211 

I.  Renal  Hyperemia. — Active  hyperemia  is  usually  an 
early  stage  of  acute  nephritis,  but  may  occur  independ- 


Fig.  63. — Sediment  from  acute  hemorrhasic  nephritis:  Red  blood -corpuscles;  leukocytes; 
renal  cells  not  fattily  degenerated;  epithelial  and  blood  casts  (Jakob). 


Fig.  64. — Sediment  from  chronic  parenchymatous  nephritis:  Hyaline  (with  cells  at- 
tached), waxy,  brown  granular,  fatty,  and  epithelial  casts;  fattily  degenerated  renal  cells, 
and  a  few  white  and  red  blood -corpuscles  (JakoJa). 

ently  as  a  result  of  temporary  irritation.  The  urine  is 
generally  decreased  in  quantity,  highly  colored,  and 
strongly  acid.     Albumin  is  always  present — usually  in 


212  THE   URINE 

traces  only,  but  sometimes  in  considerable  amount  for  a 
day  or  two.  The  sediment  contains  a  few  hyaline  and 
finely  granular  casts  and  an  occasional  red  blood-cell. 
In  very  severe  hyperemia  the  urine  approaches  that  of 
acute  nephritis. 

Passive  hyperemia  occurs  most  commonly  in  diseases 
of  the  heart  and  in  pregnancy.  The  quantity  of  urine 
is  somewhat  low  and  the  color  high,  except  in  preg- 
nancy. Albumin  is  present  in  small  amount  only.  As 
the  liver  is  usually  deranged  in  these  cases,  small  or 
moderate  amounts  of  urobilin  may  be  found.  The 
sediment  contains  a  very  few  hyaline  or  finely  granular 
casts.  In  pregnancy  the  amount  of  albumin  should  be 
carefully  watched,  as  any  considerable  quantity,  and 
especially  a  rapid  increase,  strongly  suggests  approach- 
ing eclampsia. 

2.  Nephritis. — The  various  degenerative  and  inflam- 
matory conditions  grouped  under  the  name  of  nephritis 
have  certain  features  in  common.  The  urine  in  all 
cases  contains  albumin  and  tube-casts,  and  in  all  well- 
marked  cases  shows  a  decrease  of  normal  solids,  especially 
of  urea  and  the  chlorids.  In  chronic  nephritis,  especially 
of  the  interstitial  type,  there  may  be  remissions  during 
which  the  urine  is  practically  normal.  The  degree  of 
functional  derangement  is  probably  best  ascertained  by 
the  phenol-sulphonephthalein  test  (see  p.  98).  The 
characteristics  of  the  different  forms  are  well  shown  in 
the  table  on  page  213,  modified  from  Hill. 

3.  Renal  Tuberculosis. — The  urine  is  pale,  usually 
cloudy.  The  quantity  may  not  be  affected,  but  is  apt  to 
be  increased.  In  early  cases  the  reaction  is  faintly  acid 
and  there  are  traces  of  albumin  and  a  few  renal  cells. 


THE   URINE   EST   DISEASE  213 

•  s:  o  o  B-"  g 

a  ^  3.X3  -S 


(S 


e 


p 


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214  THE    UEINE 

In  advanced  cases  the  urine  is  alkaline,  has  an  offensive 
odor,  and  is  irritating  to  the  bladder.  Albumin  in  vary- 
ing amounts  is  always  present.  Pus  is  nearly  always 
present,  though  frequently  not  abundant.  It  is  generally 
intimately  mixed  with  the  urine,  and  does  not  settle  so 
quickly  as  the  pus  of  cystitis.  Casts,  though  present,  are 
rarely  abundant,  and  are  obscured  by  the  pus.  Small 
amounts  of  blood  are  common.     Tubercle  bacilli  are 


Fig.  65. — Sediment  from  calculous  pyelitis:  Numerous  pus-corpuscles,  red  blood-cor- 
puscles, and  caudate  and  irregular  epithelial  cells;  a  combination  of  hyaline  and  pus 
casts,  and  a  few  uric-acid  crystals  (Jacol)). 

nearly  always  present,  although  animal  inoculation  may 
be  necessary  to  detect  them. 

4.  Renal  Calculus. — The  urine  is  usually  somewhat 
concentrated,  with  high  color  and  strongly  acid  reaction. 
Small  amounts  of  albumin  and  a  few  casts  may  be  pres- 
ent as  a  result  of  kidney  irritation.  Blood  is  frequently 
present,  especially  in  the  daytime  and  after  severe  ex- 
ercise. Crystals  of  the  substance  composing  the  cal- 
culus— uric   acid,   calcium   oxalate,   cystin — may   often 


THE   UEINE   IN    DISEASE  21$ 

be  found.  The  presence  of  a  calculus  generally  produces 
pyelitis,  and  variable  amounts  of  pus  then  appear,  the 
urine  remaining  acid  in  reaction. 

5.  Pyelitis. — In  pyelitis  the  urine  is  slightly  acid,  and 
contains  a  small  or  moderate  amount  of  pus,  together 
with  many  spindle  and  caudate  epithelial  cells.  Pus- 
casts  may  appear  if  the  process  extends  up  into  the  kid- 
ney tubules  (Fig.  65).  Albumin  is  always  present, 
and  its  amount,  in  proportion  to  the  amount  of  pus,  is 
decidedly  greater  than  is  found  in  cystitis.  This  fact  is 
of  much  value  in  differential  diagnosis.  Even  when  pus 
is  scanty,  albumin  is  rarely  under  0.15  per  cent.,  which 
is  the  maximum  amount  found  in  cystitis  with  abundant 
pus. 

6.  Cystitis. — In  acute  and  subacute  cases  the  urine  is 
acid  and  contains  a  variable  amount  of  pus,  with  many 
epitheUal  cells  from  the  bladder — chiefly  large  round, 
pyriform,  and  rounded  squamous  cells.  Red  blood- 
corpuscles  are  often  numerous. 

In  chronic  cases  the  urine  is  generally  alkaline.  It  is 
pale  and  cloudy  from  the  presence  of  pus,  which  is  abun- 
dant and  settles  readily  into  a  viscid  sediment.  The 
sediment  usually  contains  abundant  amorphous  phos- 
phates and  crystals  of  triple  phosphate  and  ammonium 
urate.  Vesical  epithehum  is  common.  Numerous  bac- 
teria are  always  present  (Fig.  66). 

7.  Vesical  Calculus,  Tumors,  and  Tuberculosis. — 
These  conditions  produce  a  chronic  cystitis,  with  its 
characteristic  urine.  Blood,  however,  is  more  frequently 
present  and  more  abundant  than  in  ordinary  cystitis. 
With  neoplasms,  especially,  considerable  hemorrhages 
are  apt  to  occur.    Particles  of  the  tumor  are  sometimes 


2l6  THE   URINE 

passed  with  the  urine.  No  diagnosis  can  be  made  from 
the  presence  of  isolated  tumor  cells.  In  tuberculosis 
tubercle  bacilli  can  generally  be  detected. 

8.  Diabetes  Insipidus. — Characteristic  of  this  disease 
is  the  continued  excretion  of  very  large  quantities  of  pale, 
watery  urine,  containing  neither  albumin  nor  sugar. 
The  specific  gravity  varies  between  i.ooi  and  1.005. 
The  daily  output  of  solids,  especially  urea,  is  increased. 


Pig.  66. — Sediment  from  cystitis  (chronic):  Numerous  pus-corpuscles,  epithelial  cells 
from  the  bladder,  and  bacteria;  a  few  red  blood -corpuscles  and  triple  phosphate  and 
ammonium  urate  crystals  (Jakob). 


9.  Diabetes  Mellitus. — The  quantity  of  urine  is  very 
large.  The  color  is  generally  pale,  while  the  specific 
gravity  is  nearly  always  high — 1.030  to  1.050,  very 
rarely  below  1.020.  Sometimes  in  mild  or  early  cases 
the  urine  varies  little  from  the  normal  in  quantity,  color, 
and  specific  gravity.  The  persistent  presence  of  glucose 
is  the  essential  feature  of  the  disease.  The  amount  of 
glucose  may  be  small,  but  is  often  very  great,  sometimes 
exceeding  8  per  cent.,  while  the  total  elimination  may 


THE   ITRINE  IN   DISEASE  217 

exceed  500  gm.  in  twenty-four  hours.  It  may  be  absent 
temporarily.  Acetone  is  generally  present  in  advanced 
cases.  Diacetic  and  oxybutyric  acids  may  be  present, 
and  usually  warrant  an  unfavorable  prognosis.  Accom- 
panying the  acidosis  there  is  a  corresponding  increase 
in  amount  of  ammonia. 


CHAPTER  III 

THE  BLOOD 

Preliminary  Considerations. — The  blood  consists  of 
a  fluid  of  complicated  and  variable  composition,  the 
plasma,  in  which  are  suspended  great  numbers  of  micro- 
scopic structures:  viz.,  red  corpuscles,  white  corpuscles, 
blood-platelets,  and  blood-dust. 

Red  corpuscles,  or  erythrocytes,  appear  as  biconcave 
disks,  red  when  viewed  by  reflected  light  or  in  thick  layer, 
and  straw  colored  when  viewed  by  transmitted  light  or 
in  thin  layer.  They  give  the  blood  its  red  color.  They 
are  cells  which  have  been  highly  dififerentiated  for  the 
purpose  of  carrying  oxygen  from  the  lungs  to  the  tissues. 
This  is  accompHshed  by  means  of  an  iron-bearing  pro- 
tein, hemoglobin,  which  they  contain.  In  the  lungs 
hemoglobin  forms  a  loose  combination  with  oxygen, 
which  it  readily  gives  up  when  it  reaches  the  tissues. 
Normal  erythrocytes  do  not  contain  nuclei.  They  are 
formed  from  preexisting  nucleated  cells  in  the  bone- 
marrow. 

White  corpuscles,  or  leukocytes,  are  less  highly  differ- 
entiated cells.  There  are  several  varieties.  They  all 
contain  nuclei,  and  most  of  them  contain  granules  which 
vary  in  size  and  staining  properties.  They  are  formed 
chiefly  in  the  bone-marrow  and  lymphoid  tissues. 

Blood-platelets,  or  blood- plaques,  are  colorless  or  slightly 
bluish,  spheric  or  ovoid  bodies,  usually  about  one-third  or 

•218 


SERUM  219 

one-half  the  diameter  of  an  erythrocyte.  Their  structure, 
nature,  and  origin  have  not  been  definitely  determined. 

The  blood-dust  of  Miiller  consists  of  fine  granules 
which  have  vibratory  motion.  Little  is  known  of  them. 
It  has  been  suggested  that  they  are  granules  from  dis- 
integrated leukocytes. 

The  total  amount  of  blood  is  usually  given  as  one- 
thirteenth  of  the  body  weight,  but  more  recent  investi- 
gations indicate  that  it  averages  about  one-twentieth. 

The  reaction  is  alkaline  to  Utmus. 

The  color  is  due  to  the  presence  of  hemoglobin  in  the 
red  corpuscles,  the  difference  between  the  bright  red  of 
arterial  blood  and  the  purplish  red  of  venous  blood  de- 
pending upon  the  relative  proportions  of  oxygen  and 
carbon  dioxid.  The  depth  of  color  depends  upon  the 
amount  of  hemoglobin.  In  very  severe  anemias  the 
blood  may  be  so  pale  as  to  be  designated  as  "watery." 
The  formation  of  carbon-monoxid-hemoglobin  in  coal- 
gas-poisoning  gives  the  blood  a  bright  cherry-red  color; 
while  formation  of  methemoglobin  in  poisoning  with 
potassium  chlorate  and  certain  other  substances  gives 
a  chocolate  color. 

The  clear,  pale,  straw-colored  fluid  which  remains 
after  coagulation  (see  p.  221)  and  separation  of  the  clot 
is  called  serum.  In  the  serum  are  found  the  numerous 
substances  which  the  tissues  elaborate  for  protection 
against  bacterial  and  other  harmful  agents.  In  most 
cases  these  substances,  or  "antibodies,"  are  elaborated 
only  when  the  harmful  agent  is  present  in  the  body, 
and  they  are  "specific,"  that  is,  they  are  effective  only 
against  the  one  disease  which  has  called  them  forth.  A 
test  for  the  presence  of  the  antibody  is,  therefore,  a 


220  THE   BLOOD 

test  for  the  existence  of  the  particular  disease.  The 
various  tests  based  upon  these  principles  have  within 
recent  years  become  a  very  important  part  of  clinical 
laboratory  work.  They  are  discussed  in  the  chapter 
upon  Serodiagnostic  Methods. 

The  viscosity  of  the  blood  has  received  considerable 
attention  within  recent  years.  It  is  evident  that  varia- 
tions must  markedly  influence  the  load  carried  by  the 
heart,  but  estimations  of  viscosity  have  so  far  proved 
of  comparatively  little  clinical  importance.  Compared 
with  distilled  water,  the  normal  viscosity  is  4.5.  It  is 
reduced  in  primary  and  secondary  anemia  (roughly 
proportional  to  the  grade  of  anemia),  nephritis,  cardiac 


Fig.  67. — Daland's  blood-lancet. 

lesions  with  edema,  and  usually  in  leukemia  and  malaria. 
It  is  increased  in  polycythemia,  diabetes  melhtus, 
icterus,  and  usually  in  pneumonia.  Measurement  of 
viscosity  is  simple.  The  Hess  instrument  is  one  of  the 
best.    It  is  accompanied  by  directions  for  use. 

Obtaining  the  Blood. — For  most  clinical  examinations 
only  one  drop  of  blood  is  required.  This  may  be  obtained 
from  the  lobe  of  the  ear,  the  palmar  surface  of  the  tip  of 
the  finger,  or,  in  the  case  of  infants,  the  plantar  surface 
of  the  great  toe.  In  general,  the  finger  will  be  found  most 
convenient.  With  nervous  children  the  lobe  of  the  ear 
is  preferable,  as  it  is  less  sensitive  and  its  situation  pre- 
vents their  seeing  what  is  being  done.  An  edematous 
or  congested  part  should  be  avoided;  also  a  cold,  appar- 


COAGULATION  221 

ently  bloodless  one.  The  site  should  be  well  rubbed  with 
alcohol  to  remove  dirt  and  epithelial  debris  and  to  in- 
crease the  amount  of  blood  in  the  part.  After  allowing 
sufficient  time  for  the  circulation  to  equalize,  the  skin  is 
punctured  with  a  blood-lancet  (of  which  there  are  sev- 
eral patterns  upon  the  market)  or  some  substitute,  as  a 
Hagedorn  needle,  aspirating  needle,  trocar,  a  spicule  of 
glass,  or  a  pen  with  one  of  its  nibs  broken  off.  Nothing 
is  more  unsatisfactory  than  an  ordinary  round  sewing- 
needle.  The  lancet  should  be  cleaned  with  alcohol  before 
and  after  using,  but  need  not  be  sterilized.  It  must  be 
very  sharp.  If  the  puncture  be  made  with  a/fw,  quick, 
rebounding  stroke,  at  not  too  great  a  distance,  it  is  prac- 
tically painless.  The  first  drop  of  blood  which  appears 
should  be  wiped  away,  and  the  second  used  for  exami- 
nation. The  blood  should  not  be  pressed  out,  since  this 
dilutes  it  with  serum  from  the  tissues;  but  moderate 
pressure  some  distance  above  the  puncture  is  allowable. 

When  a  larger  amount  of  blood  is  required,  it  may  be 
obtained  with  a  sterile  hypodermic  syringe  from  one  of 
the  veins  at  the  elbow,  as  described  on  p.  293. 

CHnical  study  of  the  blood  may  be  discussed  under  the 
following  heads:  I.  Coagulation.  II.  Hemoglobin.  III. 
Enumeration  of  erythrocytes.  IV.  Color  index.  V. 
Volume  index.  VI.  Enumeration  of  leukocytes.  VII. 
Enumeration  of  plaques.  VIII.  Study  of  stained  blood. 
DC.  Blood  parasites.  X.  Tests  for  recognition  of  blood. 
XL  Special  blood  pathology. 

L  COAGULATION 

Coagulation  consists  essentially  in  the  transformation 
of  fibrinogen,  one  of  the  proteins  of  the  blood-plasma, 


222 


THE  BLOOD 


into  fibrin  by  means  of  a  ferment  called  thrombin.  The 
presence  of  calcium  salts  is  necessary.  The  exact  nature 
of  the  process  is  still  undetermined.  The  resulting 
coagulum  is  made  up  of  a  meshwork  of  fibrin  fibrils  with 
entangled  corpuscles  and  platelets.  The  clear,  straw- 
colored  fluid  which  is  left  after  separation  of  the  coagu- 
lum is  called  blood-serum.  Normally, 
coagulation  takes  place  in  two  to  eight 
minutes  after  the  blood  leaves  the  ves- 
sels. The  time  is  affected  by  the  tem- 
perature, the  size  of  the  drop,  cleanliness 
of  the  instruments,  and  other  factors. 
Pathologically,  it  is  delayed  in  hemo- 
phiHa,  purpura,  scurvy,  and  icterus.  In 
treatment,  calcium  salts,  especially  the 
lactate  and  acetate,  are  used  to  hasten 
coagulation;  citric  acid,  to  retard  it. 

For  certain  purposes,  notably  in  bac- 
teriologic  and  opsonic  work,  it  is  desir- 
able to  prevent  coagulation  of  blood 
which  has  been  withdra\vn.  This  may  be 
accomplished  by  receiving  it  directly  into 
a  solution  of  i  per  cent,  sodium  citrate 
(or  ammonium  oxalate)  in  normal  salt 
solution.  This  precipitates  the  calcium 
salts  which  are  necessary  to  coagulation. 
The  simplest  method  of  ascertaining,  the  coagulation 
time  is  to  receive  several  drops  of  blood  (well  rounded 
drops  4  to  5  mm.  in  diameter)  on  a  clean  slide  and  to 
draw  a  needle  through  one  or  another  of  them  at  one- 
minute  intervals.  When  the  clot  is  dragged  along  by  the 
needle,  coagulation  has  taken  place.    Duke  uses  a  glass 


Fig.  68. — Show- 
ing difference  in 
shape  of  blood- 
drops  before  and 
after  coagulation 
(Duke's  method). 


COAGULATION 


223 


slide  to  which  two  glass  disks  5  mm.  in  diameter  are 
cemented.  Well-rounded  drops  of  blood  are  received 
on  the  disks  and  the  slide  is  inverted  across  the  top  of 
a  glass  or  beaker  containing  water  at  40°  C.  and  covered 
with  a  towel.  Coagulation  is  judged  by  the  shape  of 
the  drop  when  the  slide  is  held  in  a  vertical  position 
(Fig.  68). 

For  more  accurate  work  the  method  of  Russell  and 
Brodie  as  modified  by  Boggs  is  now  generally  used. 

Boggs'  Method. — ^The  instrument  is  shown  in  Fig.  69. 
The  bottom  of  the  box  (A)  and  the  cone  (B)  are  of  glass. 


Fig.  69. — Boggs'  coagulation  instrument:  A,  moist  chamber;  B,  glass  cone;  C,  tube  through 
which  air  is  blown. 


The  instrument  must  be  absolutely  clean.  Obtain  the 
blood  from  a  freely  flowing  puncture.  When  a  large  drop  has 
formed,  touch  the  small  end  of  the  cone  to  its  surface. 
Quickly  invert  the  cone  into  the  box.  Place  the  instrument 
on  the  microscope  and  blow  puffs  of  air  against  the  drop  of 
blood  at  intervals  by  means  of  a  rubber  bulb  attached  to 
C,  meanwhile  watching  the  motion  of  the  corpuscles  with  a 
low  power  of  the  microscope.    Coagulation  has  occurred  when 


224 


THE  BLOOD 


the  corpuscles  move  en  masse  in  a  radial  direction  and  spring 
back  to  their  original  position  (Fig.  70,  D).     The  time  is 


Fig.  70. — Diagram  showing  the  direction  taken  by  red  corpuscles  in  Boggs'  method  for 
coagulation  time:  Radial  movement  of  the  corpuscles,  D,  indicates  the  end-point  (after 
Boggs). 

counted  from  the  first  appearance  of  the  blood  from  the 
puncture  to  the  end-point. 

n.  HEMOGLOBIN 

Hemoglobin  is  an  iron-bearing  protein.  It  makes  up 
about  14  per  cent,  of  the  total  weight  of  the  blood. 
Normally,  it  is  found  only  within  the  red  corpuscles. 
When  it  is  dissolved  out  of  these  cells  and  appears  in 
the  plasma,  the  condition  is  known  as  hemoglobinemia. 


HEMOGLOBIN  225 

The  actual  amount  of  hemoglobin  is  never  estimated 
clinically:  it  is  the  relation  which  the  amount  present 
bears  to  the  normal  which  is  determined.  Thus  the  ex- 
pression, ''50  per  cent,  hemoglobin,"  when  used  clinically, 
means  that  the  blood  contains  50  per  cent,  of  the  nor- 
mal. Theoretically,  the  normal  would  be  100  per  cent., 
but  with  the  methods  of  estimation  in  general  use  the 
blood  of  healthy  persons  ranges  from  85  to  105  per  cent.; 
these  figures  may,  therefore,  be  taken  as  normal. 

Increase  of  hemoglobin,  or  hyperchromemia,  is  un- 
common, and  is  probably  more  apparent  than  real.  It 
accompanies  an  increase  in  number  of  erythrocytes,  and 
may  be  noted  in  change  of  residence  from  a  lower  to  a 
higher  altitude;  in  poorly  compensated  heart  disease 
with  cyanosis;  in  concentration  of  the  blood  from  any 
cause,  as  the  severe  diarrhea  of  cholera,  and  in  "idio- 
pathic polycythemia." 

Decrease  of  hemoglobin,  or  oligochromemia,  is  very 
common  and  important.  It  is  the  distinctive  and  most 
striking  feature  of  the  anemias  (see  p.  309) .  In  secondary 
anemia  the  hemoglobin  loss  may  be  slight  or  very  great. 
In  mild  cases  a  slight  decrease  of  hemoglobin  is  the  only 
blood  change  noted.  In  very  severe  cases,  especially  in 
repeated  hemorrhages,  malignant  disease,  and  infection 
by  the  hookworm  and  Dibothriocephalus  latus,  hemo- 
globin may  fall  to  15  per  cent.  Hemoglobin  is  always 
diminished,  and  usually  very  greatly,  in  chlorosis  (aver- 
age about  40  to  45  per  cent.),  pernicious  anemia  (average 
about  20  to  25  per  cent.),  and  leukemia  (usually  about  40 
to  50  per  cent.). 

Estimation  of  hemoglobin  is  less  tedious  and  usually 
more  helpful   than   a  red   corpuscle   count.     It  offers 

15 


226  THE    BLOOD 

the  simplest  and  most  certain  means  of  detecting  the 
existence  and  degree  of  anemia,  and  of  judging  the  effect 
of  treatment  in  anemic  conditions.  Pallor,  observed 
clinically,  does  not  always  denote  anemia. 

There  are  many  methods,  but  none  is  entirely  satis- 
factory. Those  which  are  most  widely  used  are  here 
described: 

(i)  Von  Fleischl  Method. — ^The  apparatus  consists  of  a 
stand  somewhat  like  the  base  and  stage  of  a  microscope 


Fig.  71. — Von  Fleischrs  hemoglobinoraeter:  a,  Stand;  b,  narrow  wedge-shaped  piece 
of  colored  glass  fitted  into  a  frame  (f).  which  passes  under  the  chamber;  d,  hollow  metal 
cylinder,  diNnded  into  two  compartments,  which  holds  the  blood  and  water;  e.  plaster-of- 
Paris  plate  from  which  the  light  is  reflected  through  the  chamber;  /,  screw  by  which  the 
frame  containing  the  graduated  colored  glass  is  moved;  g.  capillary  tube  to  collect  the 
blood;  h,  pipet  for  adding  the  water;  1,  opening  through  which  may  l)e  seen  the  scale 
indicating  percentage  of  hemoglobin. 


(Fig.  71).  Under  the  stage  is  a  movable  bar  of  colored  glass, 
shading  from  pale  pink  at  one  end  to  deep  red  at  the  other. 
The  frame  in  which  this  bar  is  held  is  marked  with  a  scale  of 


HEMOGLOBIN  227 

hemoglobin  percentages  corresponding  to  the  different  shades 
of  red.  By  means  of  a  rack  and  pinion  the  color-bar  can  be 
moved  from  end  to  end  beneath  a  round  opening  in  the  center 
of  the  stage.  A  small  metal  cylinder,  which  has  a  glass  bot- 
tom and  which  is  divided  vertically  into  two  equal  compart- 
ments, can  be  placed  over  the  opening  in  the  stage  so  that  one 
of  its  compartments  lies  directly  over  the  color-bar.  Accom- 
panying the  instrument  are  a  number  of  short  capillary  tubes 
in  metal  handles. 

Having  punctured  the  finger-tip  or  lobe  of  the  ear,  as  al- 
ready described,  wipe  off  the  first  drop  of  blood,  and  from  the 
second  fill  one  of  the  capillary  tubes.  Hold  the  tube  hori- 
zontally, and  touch  its  tip  to  the  drop  of  blood,  which  will 
readily  flow  into  it  if  it  be  clean  and  dry.  Avoid  getting  any 
blood  upon  its  outer  surface.  With  a  medicme- dropper  rinse 
the  blood  from  the  tube  into  one  of  the  compartments  of  the 
cylinder,  using  distilled  water,  and  mix  well.  Fill  both  com- 
partments level  full  with  distilled  water,  and  place  the  cylin- 
der over  the  opening  in  the  stage,  so  that  the  compartment 
which  contains  only  water  lies  directly  over  the  bar  of  colored 
glass.  If  there  are  any  clots  in  the  hemoglobin  compart- 
ment, clean  the  instrument  and  begin  again. 

In  a  dark  room,  with  the  light  from  a  candle  reflected  up 
through  the  cylinder,  move  the  color-bar  along  with  a  jerking 
motion  until  both  compartments  have  the  same  depth  of  color. 
The  number  upon  the  scale  corresponding  to  the  portion  of  the 
color-bar  which  is  now  under  the  cylinder  gives  the  percent- 
age of  hemoglobin.  While  comparing  the  two  colors,  place 
the  instrument  so  that  they  will  fall  upon  the  right  and  left 
halves  of  the  retina,  rather  than  upon  the  upper  and  lower 
halves;  and  protect  the  eye  from  the  light  with  a  cylinder  of 
paper  or  pasteboard.  After  use,  clean  the  metal  cylinder  with 
"water,  and  wash  the  capillary  tube  with  water,  alcohol,  and 
ether,  successively.  Results  with  this  instrument  are  accu- 
rate to  within  about  5  per  cent. 


228  THE   BLOOD 

(2)  The  Fleischl-Miescher  instrument,  a  modification  of 
the  preceding,  is  generally  considered  the  most  accurate 
hemoglobinometer  available.  It  is,  however,  better  adapted 
to  laboratory  use  than  to  the  needs  of  the  clinician.  Detailed 
instructions  accompany  each  instrument.  The  chief  differ- 
ences from  the  von  Fleischl  are:  (i)  The  blood  is  more  accu- 
rately measured  and  diluted,  a  pipet  like  that  accompanying 
the  hemocytometer  being  used;  (2)  o.i  per  cent,  solution  of 
sodium  carbonate  is  used  instead  of  water  for  diluting;  (3) 
the  glass  bar  is  more  accurately  colored;  (4)  there  are  two 
cylindric  cells,  one  four-fifths  the  depth  of  the  other;  and  (5) 
the  cell  is  covered  with  a  glass  disk  and  a  metal  cap  with 
a  slit  through  which  the  reading  is  made. 

(3)  The  Sahli  hemoglobinometer  (Fig.  72)  is  an  improved 
form  of  the  well-known  Gowers  instrument.  It  consists  of  a 
hermetically  sealed  comparison  tube  containing  a  suspension 
of  acid  hematin,  a  graduated  test-tube  of  the  same  diameter, 
and  a  pipet  of  20-c.mm.  capacity.  The  two  tubes  are  held 
in  a  black  frame  with  a  white  ground-glass  back. 

Place  decinormal  hydrochloric  acid  solution  in  the  gradu- 
ated tube  to  the  mark  10.  Obtain  a  drop  of  blood  and  draw 
it  into  the  pipet  to  the  20  c.mm.  mark.  Wipe  off  the  tip  of  the 
pipet,  blow  its  contents  into  the  hydrochloric  acid  solution  in 
the  tube,  and  rinse  well.  The  hemoglobin  is  changed  to  acid 
hematin.  Place  the  two  tubes  in  the  compartments  of  the 
frame;  let  stand  one  minute;  and  dilute  the  fluid  with  water 
drop  by  drop,  mixing  after  each  addition,  until  it  has  exactly 
the  same  color  as  the  comparison  tube.  The  graduation  cor- 
responding to  the  surface  of  the  fluid  then  indicates  the  per- 
centage of  hemoglobin.  Mixing  may  be  done  by  closing  the 
tube  with  the  finger  and  inverting,  but  care  should  be  exer- 
cised to  see  that  none  of  the  fluid  is  removed  by  adhering  to 
the  finger.  Decinormal  hydrochloric  acid  solution  may  be 
prepared  with  sufficient  accuracy  for  this  purpose  by  adding 


HEMOGLOBIN 


229 


15  c.c.  of  the  concentrated  acid  to  985  c.c.  distilled  water. 
A  little  chloroform  should  be  added  as  a  preservative. 

This  method  is  very  satisfactory  in  practice,  and  is  accurate 
to  within  5  per  cent.  Unfortunately,  not  all  the  instruments 
upon  the  market  are  well  standardized,  and  the  comparison 


Fig.  72. — Sahli's  hemoglobinometer. 


tube  does  not  keep  its  color  unchanged  indefinitely.  Usually, 
however,  the  apparent  fading  is  due  to  the  fact  that  the 
hematin  is  in  suspension  and  may  settle  out  if  the  instru- 
ment lies  unused  for  some  time.  This  can  be  remedied  by 
inverting  the  tube  a  number  of  times.  Most  tubes  contain 
a  glass  bead  to  facilitate  mixing. 


230  THE    BLOOD 

(4)  Dare's  hemoglobinometer  (Fig.  73)  differs  from  the 
others  in  using  undiluted  blood.  The  blood  is  allowed  to  flow 
by  capillarity  into  the  slit  between  two  small  plates  of  glass. 
It  is  then  placed  in  the  instrument  and  compared  with  differ- 
ent portions  of  a  circular  disk  of  colored  glass.  The  reading 
must  be  made  quickly,  before  clotting  takes  place.  This 
instrument  is  easy  to  use,  and  is  one  of  the  most  accurate. 


F'K-  73' — Dare's  hemoglobinometer. 

(5)  Hammerschlag  Method. — ^This  is  an  indirect  method 
which  depends  upon  the  fact  that  the  percentage  of  hemo- 
globin varies  directly  with  the  specific  gravity  of  the  blood. 
It  yields  fairly  accurate  results  except  in  leukemia,  where  the 
large  number  of  leukocytes  disturbs  the  relation,  and  in 
dropsical  conditions. 

MLx  chloroform  and  benzol  in  a  urinometer  tube,  so  that 
the  specific  gravity  of  the  mixture  is  near  the  probable  specific 
gravity  of  the  blood.  Add  a  drop  of  blood  by  means  of  a 
pipet  of  small  caliber.  A  pipet  like  that  shown  in  Fig.  169,  A, 
will  be  found  satisfactory.  If  the  drop  floats  near  the  surface, 
add  a  little  benzol;  if  it  sinks  to  the  bottom,  add  a  little  chloro- 
form.   When  it  remains  stationary  near  the  middle,  the  mix- 


HEMOGLOBIN 


231 


ture  has  the  same  specific  gravity  as  the  blood.  Take  the 
specific  gravity  with  a  urinometer,  and  obtain  the  correspond- 
ing percentage  of  hemoglobin  from  the  following  table: 


Specific 
Gravity. 


Hemoglobin 
Per  Cent. 


Specific 
Gravity. 


Hemoglobin 
Per  Cent. 


I.033-I.035 25-30 

I.035-I.038 30-35 

I.038-1.040 35-40 

I.040-I.045 40-45 

I.045-I.048— 45-55 


I.048-I.050 55-65 

I.050-I.053 65-70 

I.053-I.055 70-75 

I.055-I.057 75-85 

I.057-I.060 85-95 


For  accurate  results  with  this  method,  care  and  patience 
are  demanded.    The  following  precautions  must  be  observed: 


Fig  74. — Tallqvist's  hemoglobin  scale. 


-  (a)  The  two  fluids  must  be  well  mixed  after  each  addition 
of  chloroform  or  benzol.  Close  the  tube  with  the  thumb  and 
invert  several  times.    Should  this  cause  the  drop  of  blood  to 


232  THE   BLOOD 

break  up  into  very  small  ones,  adjust  the  specific  gravity  as 
accurately  as  possible  with  these,  and  test  it  with  a  fresh  drop. 

(b)  The  drop  of  blood  must  not  be  too  large;  it  must  not 
contain  an  air-bubble,  it  must  not  adhere  to  the  side  of  the 
tube,  and  it  must  not  remain  long  in  the  fluid. 

(c)  The  urinometer  must  be  standardized  for  the  chloro- 
form-benzol mixture.  Most  urinometers  give  a  reading  two 
or  three  degrees  too  high,  owing  to  the  low  surface  tension. 
Make  a  mbcture  such  that  a  drop  of  distilled  water  will  re- 
main suspended  in  it  {i.  c,  with  a  specific  gravity  of  i.cxx)) 
and  correct  the  urinometer  by  this. 

(6)  Tallqvist  Method. — The  popular  Tallqvist  hemo- 
globinometer  consists  simply  of  a  book  of  small  sheets  of  ab- 
sorbent paper  and  a  carefully  printed  scale  of  colors  (Fig.  74). 

Take  up  a  large  drop  of  blood  with  the  absorbent  paper, 
and  when  the  humid  gloss  is  leaving,  before  the  air  has  dark- 
ened the  hemogobin,  compare  the  stain  with  the  color  scale. 
The  color  which  it  matches  gives  the  percentage  of  hemo- 
globin. Except  in  practised  hands,  this  method  is  accurate 
only  to  within  10  or  20  per  cent. 

Of  the  methods  given,  the  physician  should  select  the 
one  which  best  meets  his  needs.  With  any  method, 
practice  is  essential  to  accuracy.  The  von  Fleischl  was  for 
many  years  the  standard  instrument,  but  is  now  little 
used.  For  accurate  work  the  best  instruments  are  the 
von  Fleischl-Miescher  and  the  Dare.  The  former  is 
essentially  a  laboratory  instrument.  The  Dare  is  easy 
to  use  and  to  clean,  and  is  probably  the  best  for  clinical 
work.  The  Sahli,  although  less  easy  to  use  and  prob- 
ably less  accurate,  is  inexpensive  and  is  very  satisfac- 
tory, provided  a  well-standardized  color-tube  is  obtained. 
The  specific  gravity  method  is  useful  when  special  in- 
struments are  not  at  hand.     The  Tallqvist  scale  is  so 


ENUMERATION   OF   ERYTHROCYTES  233 

inexpensive  and  so  convenient  that  it  should  be  used  by 
every  physician  at  the  bedside  and  in  hurried  office 
work;  but  it  should  not  supersede  the  more  accurate 
methods. 

m.  ENUMERATION  OF  ERYTHROCYTES 

In  health  there  are  about  5,000,000  red  corpuscles  per 
cubic  millimeter  of  blood.  Normal  variations  are  slight. 
The  number  is  generally  a  little  less — about  4,500,000 — 
in  women.  Hawk  finds  the  normal  for  athletes  in  train- 
ing to  be  5,500,000. 

Increase  of  red  corpuscles,  or  polycythemia,  is  unimpor- 
tant. There  is  a  decided  increase  following  change  of 
residence  from  a  lower  to  a  higher  altitude,  averaging 
about  50,000  corpuscles  for  each  1000  feet,  but  frequently 
much  greater.  The  increase,  however,  is  not  permanent. 
In  a  few  months  the  erythrocytes  return  to  nearly  their 
original  number.  Three  views  are  offered  in  explanation : 
(a)  Concentration  of  the  blood,  owing  to  increased  evap- 
oration from  the  skin;  {h)  stagnation  of  corpuscles  in 
the  peripheral  vessels  because  of  lowered  blood-pressure; 
(c)  new  formation  of  corpuscles,  this  giving  a  compensa- 
tory increase  of  aeration  surface. 

Pathologically,  polycythemia  is  uncommon.  It  may 
occur  in:  {a)  Concentration  of  the  blood  from  severe 
watery  diarrhea;  {h)  chronic  heart  disease,  especially  the 
congenital  variety,  with  poor  compensation  and  cyanosis ; 
and  (c)  idiopathic  polycythemia,  which  is  considered  to  be 
an  independent  disease,  and  is  characterized  by  cyanosis, 
blood-counts  of  7,000,000  to  10,000,000,  hemoglobin  120 
to  150  per  cent.,  and  a  normal  number  of  leukocytes. 

Decrease   of  red   corpuscles,    or   oligocythemia.     Red 


234 


THE    BLOOD 


corpuscles    and    hemoglobin   are   commonly  decreased 
together,  although  usually  not  to  the  same  extent. 

Oligocythemia  occurs  in  all  but  the  mildest  symp- 
tomatic anemias.  The  blood-count  varies  from  near  the 
normal  in  moderate  cases  down  to  1,500,000  in  very 
severe  cases.  There  is  always  a  decrease  of  red  cells  in 
chlorosis,  but  it  is  often  slight,  and  is  relatively  less  than 
the  decrease  of  hemoglobin.    Leukemia  gives  a  decided 


Fig-  7S- — Thoma-Zeiss  hemacytometer:  a.  Slide  used  in  counting;  b,  sectional  view; 
d,  red  pipet;  e,  white  pipet. 

oligocythemia,  the  average  count  being  about  3,000,000. 
The  greatest  loss  of  red  cells  occurs  in  pernicious  anemia, 
where  counts  below  i  ,000,000  are  not  uncommon. 

Method  of  Counting. — The  most  widely  used  instru- 
ment for  counting  the  corpuscles  is  that  of  Thoma-Zeiss. 
Biirker's  hemacytometer  is  more  accurate  and  has  been 
very  favorably  received.  The  new  Thoma-Metz  instru- 
ment is  convenient  for  routine  working.  The  hematocrit 
is  not  to  be  recommended  for  accuracy,  since  in  anemia. 


ENUMERATION    OF  ERYTHROCYTES 


235 


where  blood-counts  are  most  important,  the  red  cells 
vary  greatly  in  size  and  probably  also  in  elasticity.  The 
hematocrit  is,  however,  useful  in  determining  the  rela- 
tive volume  of  corpuscles  and  plasma  (see  Volume  In- 
dex, p.  245). 

The  Thoma-Zeiss  instrument  consists  of  two  pipets  for 
diluting  the  blood  and  a  counting  chamber  (Fig.  75).    The 


aUjJ 


iiok 


Fig.  76. — Thoma  rulin?  of  counting  chamber,  showing  red  corpuscles  in  left  upper  comer. 


rubber  tubes  which  come  with  the  pipets  are  too  short  and 
too  flexible  and  should  be  replaced.  For  this  purpose  nothing 
is  so  good  as  a  rubber  catheter.  The  counting  chamber  is  a 
glass  slide  with  a  square  platform  in  the  middle.  In  the 
center  of  the  platform  is  a  circular  opening,  in  which  is  set 


236 


THE   BLOOD 


a  small  circular  disk  in  such  a  manner  that  it  is  surrounded 
by  a  "ditch,"  and  that  its  surface  is  exactly  one-tenth  of  a 
millimeter  below  the  surface  of  the  square  platform.  Upon 
this  disk  is  ruled  a  square  millimeter,  subdivided  into  400 
small  squares.  Each  fifth  row  of  small  squares  has  double 
rulings  for  convenience  in  counting  (Fig.  76).  This  ruling, 
known  as  the  Thoma,  constitutes  the  central  square  milli- 
meter of  most  of  the  more  recent  forms,  such  as  the  Zappert 


Fig  77. — Method  of  drawinj<  blood  into  the  pipet  (Boston). 


and  theTurck  (see  Fig.  82).  A  thick  cover-glass,  ground  per- 
fectly plane,  accompanies  the  counting  chamber.  Ordinary 
cover-glasses  are  of  uneven  surface,  and  should  not  be  used 
with  this  instrument. 

It  is  evident  that,  when  the  cover-glass  is  in  place  upon 
the  platform,  there  is  a  space  exactly  one-tenth  of  a  milli- 
meter thick  between  it  and  the  disk;  and  that,  therefore,  the 
square  millimeter  ruled  upon  the  disk  forms  the  base  of  a 
space  holding  exactly  one-tenth  of  a  cubic  millimeter. 


ENUMERATION   OF   ERYTHROCYTES  237 

Technic. — To  count  the  red  corpuscles,  use  the  pipet  with 
1 01  engraved  above  the  bulb.  It  must  be  clean  and  dry. 
Puncture  the  skin,  wipe  off  the  first  drop  of  blood,  and  fill 
the  pipet  from  the  second,  sucking  the  blood  to  the  mark 
0.5  or  i.o,  according  to  the  dilution  desired.  While  doing  this, 
hold  the  pipet  at  nearly  right  angles  to  the  line  of  vision,  so 
that  the  exact  height  of  the  column  may  be  easUy  seen.  The 
side  of  the  tip  should  rest  against  the  skin,  but  the  end  must 
be  free.  Air-bubbles  will  enter  if  the  drop  is  too  small  or  if 
the  tip  is  not  kept  immersed.  Should  the  blood  go  slightly 
beyond  the  mark,  draw  it  back  by  touching  the  tip  of  the 
pipet  to  a  moistened  handkerchief.  Quickly  wipe  off  the 
blood  adhering  to  the  tip,  plvmge  it  into  the  diluting  fluid, 
and  suck  the  fluid  up  to  the  mark  loi,  slightly  rotating  the 
pipet  meanwhile.  This  dilutes  the  blood  i :  200  or  i :  100, 
according  to  the  amount  of  blood  taken.  Except  in  cases 
of  severe  anemia,  a  dilution  of  i  :2oo  is  preferable.  Close  the 
ends  of  the  pipet  with  the  fingers,  and  shake  vigorously  until 
the  blood  and  diluting  fluid  are  well  mixed,  keeping  the  pipet 
horizontal  meanwhile. 

When  it  is  not  convenient  to  count  the  corpuscles  at  once, 
place  a  heavy  rubber  band  aroimd  the  pipet  so  as  to  close 
the  ends,  inserting  a  small  piece  of  rubber-cloth  or  other 
tough,  non-absorbent  material,  if  necessary,  to  prevent  the 
tip  from  punching  through  the  rubber.  It  may  be  kept  thus 
for  twenty-four  hours  or  longer. 

When  ready  to  make  the  count,  clean  the  counting  chamber 
and  cover-glass,  and  place  a  sheet  of  paper  over  them  to 
keep  off  dust.  Mix  the  fluid  thoroughly  by  shaking;  blow  2 
or  3  drops  from  the  pipet,  wipe  off  its  tip,  and  then  place 
a  small  drop  (the  proper  size  can  be  learned  only  by  experi- 
ence) upon  the  disk  of  the  counting  chamber.  Adjust  the 
cover  immediately.  Hold  it  by  diagonal  corners  above  the 
drop  of  fluid  so  that  a  third  corner  touches  the  slide  and  rests 
upon  the  edge  of  the  platform.    Place  a  finger  upon  this  cor- 


238  THE    BLOOD 

ner,  and,  by  raising  the  finger,  allow  the  cover  to  fall  quickly 
into  place.  If  the  cover  be  properly  adjusted,  faint  concentric 
lines  of  the  prismatic  colors — Newton's  rings — can  be  seen 
between  it  and  the  platform  when  the  slide  is  viewed  ob- 
liquely. They  indicate  that  the  two  surfaces  are  in  close 
aj^position.  If  they  do  not  appear  at  once,  slight  pressure 
upon  the  cover  may  bring  them  out.  Failure  to  obtain  them 
is  usually  due  to  dirty  slide  or  cover — both  must  be  perfectly 
clean  and  free  from  dust.  The  drop  placed  upon  the  disk 
must  be  of  such  size  that,  when  the  cover  is  adjusted,  it 
nearly  or  quite  covers  the  disk,  and  that  none  of  it  runs  over 
into  the  "ditch."  There  should  be  no  bubbles  upon  the  ruled 
area. 

The  following  is  a  somewhat  easier  method  of  applying  the 
cover:  Place  a  drop  of  fluid  upon  the  ruled  disk.  The  size 
of  the  drop  is  of  no  great  consequence,  if  only  it  be  large 
enough.  Place  the  cover-glass  flat  upon  one  side  of  the  plat- 
form with  its  edge  close  to  the  drop  of  fluid,  and  hold  it 
firmly  down  with  the  two  index-fingers,  or  with  the  index- 
finger  and  middle  fingers  of  the  right  hand.  Now  slide  it 
firmly  and  quickly  into  place.  If  the  drop  of  fluid  is  too  large, 
the  excess  will  be  caught  on  the  top  of  the  cover.  A  moder- 
ately thin  cover  is  best. 

Allow  the  corpuscles  to  settle  for  a  few  minutes,  and  then 
examine  with  a  low  power  to  see  that  they  are  evenly  dis- 
tributed. If  they  are  not  evenly  distributed  over  the  whole  disk, 
the  counting  chamber  must  be  cleaned  and  a  new  drop  placed 
in  it. 

Probably  the  most  satisfactory  objective  for  counting  is  the 
8  mm.  or  the  4  mm.  with  long  working  distance.  To  under- 
stand the  principle  of  counting,  it  is  necessary  to  remember 
that  the  square  millimeter  (400  small  squares)  represents  a 
capacity  of  one-tenth  of  a  cubic  millimeter.  Find  the  num- 
ber of  corpuscles  in  the  square  millimeter,  multiply  by  10  to 
find  the  number  in  i  c.mm.  of  the  diluted  blood,  and  finally, 


ENUMERATION    OF    ERYTHROCYTES 


239 


by  the  dilution,  to  find  the  number  in  i  c.mm.  of  undiluted 
blood.  Instead  of  actually  counting  all  the  corpuscles,  it  is 
customary  to  count  those  in  only  a  limited  number  of  small 
squares,  and  from  this  to  calculate  the  number  in  the  square 
millimeter.  Nearly  every  worker  has  his  own  method  of 
doing  this.  The  essential  thing  is  to  adopt  a  method  and 
adhere  to  it. 


Fig.  78. — Appearance  of  microscopic  field  in  counting  red  corpuscles.  The  arrow  indicates 
the  squares  to  be  counted. 


In  practice  a  convenient  procedure  is  as  follows:  With  a 
dilution  of  1  :  200,  count  the  cells  in  80  small  squares,  and  to 
the  sum  add  4  ciphers;  with  dilution  of  i  :  100,  count  40  small 
squares  and  add  4  ciphers.  Thus,  if  with  i  :  200  dilution,  450 
corpuscles  were  counted  in  80  squares,  the  total  count  would 
be  4,500,000  per  c.mm.    This  method  is  sufficiently  accurate 


240  THE   BLOOD 

for  all  clinical  purposes,  provided  the  corpuscles  are  evenly 
distributed  and  3  drops  from  the  pipet  be  counted.  It  is 
convenient  to  count  a  block  of  20  small  squares,  as  indicated 
in  Fig.  78,  in  each  corner  of  the  large  square.  Four  columns 
of  5  squares  each  are  counted.  The  double  rulings  show  when 
the  bottom  of  a  column  has  been  reached  and  also  indicate 
the  fourth  column.  In  the  writer's  opinion  it  is  easier  to 
count  in  vertical  than  horizontal  rows.  If  distribution  be 
even,  the  difference  between  the  number  of  cells  in  any  two 
such  blocks  should  not  exceed  twenty.  In  order  to  avoid 
confusion  in  counting  cells  which  lie  upon  the  border-lines, 
the  following  rule  is  generally  adopted:  Corpuscles  which 
touch  the  upper  and  left  sides  should  be  counted  as  if  within  the 
squares,  those  touching  the  lower  and  right  sides,  as  outside;  and 
vice  versa. 

Diluting  Fluids. — The  most  widely  used  are  Hayem's  and 
Toisson's.  Both  of  these  have  high  specific  gravities,  so  that, 
when  well  mixed,  the  corpuscles  do  not  separate  quickly. 
Toisson's  fluid  is  probably  the  better  for  beginners,  because  it 
is  colored  and  can  easily  be  seen  as  it  is  drawn  into  the  pipet. 
It  stains  the  nuclei  of  leukocytes  blue,  but  this  is  no  real  ad- 
vantage. It  must  be  filtered  frequently  because  of  the  ready 
growth  of  fungi  in  it. 

Hayem's  Fluid.  Toisson's  Fluid. 

Mercuric  chloric! 0.5       Sodium  chlorid i.o 

Sodium  sulphate 5.0       Sodium  sulphate 8.0 

Sodium  chlorid i  .0       Glycerin 30.0 

Distilled  water 200.0       Distilled  water 160.0 

Methyl-violet,  5  B  to  give  a  strong 
purple  color. 

Sources  of  Error. — The  most  common  sources  of  error  in 
making  a  blood-count  are: 

(a)  Inaccurate  dilution,  usually  from  faulty  technic, 
occasionally  from  inaccurately  graduated  pipets.  Only  an 
instrument  of  standard  make  can  be  relied  upon. 


ENUMERATION   OF   ERYTHROCYTES  24 1 

(b)  Too  slow  manipulation,  allowing  a  little  of  the  blood 
to  coagulate  and  remain  in  the  capillary  portion  of  the 
pipet. 

(c)  Inaccuracy  in  depth  of  counting  chamber  usually  due 
to  imperfect  application  of  the  cover-glass,  but  sometimes  to 
softening  of  the  cement  by  alcohol  or  heat.  The  slide  should 
not  be  cleaned  with  alcohol  nor  left  to  Ue  in  the  warm  sun- 
shine. 

(d)  Uneven  distribution  of  the  corpuscles.  This  results 
when  the  blood  has  partially  coagulated,  when  it  is  not 
thoroughly  mixed  with  the  diluting  fluid,  or  when  the  cover- 
glass  is  not  applied  soon  enough  after  the  drop  is  placed  upon 
the  disk. 

(e)  The  presence  of  yeasts,  which  may  be  mistaken  for 
corpuscles,  in  the  diluting  fluid 

Cleaning  the  Instrument. — ^The  instrument  should  be 
cleaned  immediately  after  using,  and  the  counting  chamber 
and  cover  must  be  cleaned  again  just  before  use. 

Transfer  the  rubber  tube  to  the  small  end  of  the  pipet  and 
draw  through  it,  successively,  water,  alcohol,  ether,  and  air. 
This  can  be  done  with  the  mouth,  but  it  is  much  better  to 
use  a  rubber  bulb  or  suction  filter  pump.  When  the  mouth  is 
used,  the  moisture  of  the  breath  will  condense  upon  the 
interior  of  the  pipet  unless  the  fluids  be  shaken  and  not  blown 
out.  If  blood  has  coagulated  in  the  pipet — which  happens 
when  the  work  is  done  too  slowly — dislodge  the  clot  with  a 
horsehair,  never  w4th  a  wire,  and  clean  with  strong  sulphuric 
acid,  or  let  the  pipet  stand  over  night  in  a  test-tube  of  the 
acid.  Even  if  the  pipet  does  not  become  clogged,  it  should  be 
occasionally  cleaned  in  this  way.  When  the  etched  gradua- 
tions on  the  pipets  become  dim,  they  can  be  renewed  by 
rubbing  with  a  grease  pencil. 

"  Wash  the  counting-chamber  and  the  cover  with  water  and 
dry  them  with  clean  soft  linen.    Alcohol  may  be  used  to  clean 
the  latter,  but  never  the  former. 
16 


242 


THE   BLOOD 


BiJrker's  hemacytometer  (Fig.  79)'. — This  modification  of 
the  Thoma-Zeiss  instrument  allows  of  greater  accuracy. 
It  consists  of  a  counting  slide  with  cover-glass,  three  pipets 
— (a)  for  measuring  blood,  {b)  measuring  diluting  fluid,  and 
(f)  transferring  the  diluted  blood  to  the  slide — and  one  or 
more  small  flasks  for  mixing  blood  and  diluting  fluid. 

The  floor-piece  of  the  counting  slide,  instead  of  being  cir- 
cular, as  in  the  Thoma-Zeiss  instrument,  consists  of  a  plate 


Fig.  7Q. —  Hurk^T's  hemacytometer  with  pipets  for  counting  red-corpuscles:  A,  counting 
slide  with  cover-glass  in  place;  B.  pip)et  for  measuring  blood;  C,  pipet  for  measuring 
diluting  fluid;  D,  pipet  for  transferring  diluted  blood  to  slide;  E.  mixing  flask. 

of  glass  5  mm.  wide  and  25  mm.  long,  which  extends  across 
the  slide.  This  is  divided  across  the  middle  by  a  deep  groove 
1.5  mm.  wide,  and  upon  each  portion  is  a  ruled  area.  On 
each  side  of  the  floor-piece  and  separated  from  it  by  a  ditch 
is  a  glass  platform  o.i  mm.  higher  than  the  ruled  areas. 
When  the  cover-glass  is  adjusted  upon  the  platform,  the  ends 
of  the  floor-piece  project  beyond  it.  There  are  two  clamps 
to  hold  the  cover  in  place. 


ENUMERATION    OF   ERYTHROCYTES  243 

When  the  count  is  to  be  made,  the  correct  amount  of 
Hayem's  fluid  is  placed  in  the  mixing  flask,  and  the  blood, 
accurately  measured  in  the  blood-pipet,  is  added  and  well 
mixed,  care  being  taken  that  none  of  the  fluid  touches  the 
stopper  of  the  flask.  The  cover-glass  is  then  carefully  ad- 
justed so  as  to  show  Newton's  rings,  and  is  clamped  in  place. 
A  drop  of  the  diluted  blood  is  placed  on  each  of  the  projecting 
ends  of  the  floor-piece  by  means  of  the  special  pipet.  The 
fluid  will  run  under  the  cover  by  capillary  attraction.  The 
slide  is  now  placed  on  the  microscope  with  the  diaphragm 
wide  open  and  viewed  obliquely  with  the  imaided  eye.  If 
the  film  of  corpuscles  is  not  uniform,  the  slide  must  be  cleaned 
and  filled  again.    The  coimt  is  made  in  the  usual  way.    As 


© 


Fig.  80. — Thoma-Metz  hemacytometer  and  diagram  showing  ruling  in  ocular. 

ordinarily  supplied  the  instnmient  has  a  special  ruling,  but 
other  rulings  may  be  obtained. 

The  Biirker  counting  chamber  can  now  be  obtained  with 
the  regular  Thoma  mixing  pipets. 

Thoma-Metz  Hemacytometer  (Fig.  80). — ^This  new  in- 
strument introduces  certain  conveniences  into  the  routine 
counting  of  both  red  cells  and  leukocytes.  Its  special 
feature  is  that  the  ruling  is  engraved  upon  a  disk  in  the 
ocular  instead  of  upon  the  counting  slide.  This  disk  is  ruled 
wifh  a  large  circle  and  with  a  square,  which  in  turn  is  sub- 
divided into  four  smaller  squares. 

For  the  red  count  the  squares  are  used.  The  four  small 
squares  have  each  the  same  value  as  the  small  squares  of  the 


244  THE   BLOOD 

Thoma  ruling  (7^77  sq.  mm.),  and  the  count  may  be  conducted 
as  already  described. 

The  circle  is  used  for  counting  leukocytes.  Its  area  cor- 
responds to  one-tenth  of  a  square  millimeter  when  the  correct 
magnification  is  used.  The  leukocytes  are  counted  as  in  the 
circle  method  described  on  p.  256. 

A  decided  advantage  of  this  instnmient  is  that  the  ruled 
lines  are  always  sharp  and  clear.  The  eye-lens  of  the  ocular 
can  be  focused  to  suit  different  eyes.  The  chief  disadvantage 
and  a  source  of  inaccuracy  lies  in  the  fact  that  the  values  of 
the  ruled  areas  vary  according  to  magnification.  The  makers 
say  that  values  are  correct  as  above  given  when  the  Leitz 
No.  6  objective  (4-mm.  focus)  is  used  with  tube  length  of 
170  mm.  Slight  variations  with  other  objectives  can  be 
compensated  by  altering  the  tube  length.  For  accurate 
evaluation  a  square  is  ruled  on  the  counting  slide,  and  the 
tube  length  should  be  so  adjusted  that  this  square  exactly 
coincides  with  the  large  square  in  the  ocular. 

IV.  COLOR  INDEX 

This  is  an  expression  which  indicates  the  amount  of 
hemoglobin  in  each  red  corpuscle  compared  with  the 
normal  amount.  For  example,  a  color  index  of  i.o  in- 
dicates that  each  corpuscle  contains  the  normal  amount 
of  hemoglobin;  of  0.5,  that  each  contains  one-half  the 
normal. 

The  color  index  is  most  significant  in  chlorosis  and 
pernicious  anemia.  In  the  former  it  is  usually  much 
decreased;  in  the  latter,  generally  much  increased.  In 
symptomatic  anemia  it  is  generally  moderately  dirnin- 
ished. 

To  obtain  the  color  index,  divide  the  percentage  of  hemo- 
globin by  the  percentage  of  corpuscles.    The  percentage  of 


VOLUME   INDEX  245 

corpuscles  is  found  by  multiplying  the  first  two  figures  of  the 
red  corpuscle  count  by  2.  This  simple  method  holds  good  for 
all  coimts  of  1,000,000  or  more.  Thus,  a  coimt  of  2,500,000 
is  50  per  cent,  of  the  normal.  If,  then,  the  hemoglobin  has 
been  estimated  at  40  per  cent.,  divide  40  (the  percentage 
of  hemoglobin)  by  50  (the  percentage  of  corpuscles).  This 
gives  f ,  or  0.8,  as  the  color  index. 

V.  VOLUME  INDEX 

The  term  "volume  index"  was  introduced  by  Capps 
to  express  the  average  size  of  the  red  cells  of  an  individual 
compared  with  their  normal  size.  It  is  the  quotient 
obtained  by  dividing  the  volume  of  red  corpuscles  (ex- 
pressed in  percentage  of  the  normal)  by  the  number  of 
red  corpuscles,  also  expressed  in  percentage  of  the  nor- 
mal. 

The  volume  index  more  or  less  closely  parallels  the 
color  index,  and  variations  have  much  the  same  sig- 
nificance. The  following  are  averages  of  the  examina- 
tions reported  by  Larrabee  in  the  "Journal  of  Medical 
Research": 

Red  corpuscles  Hemoglobin  per 

per  cubic  cent,  by  Sahli  Color  Volume 

millimeter.  instrument.  index.  index. 

Normal  males 5,267,250  103.0  0.98  1.007 

Normal  females 4,968,667  106.0  1.06  i.ooi 

Primary  pernicious  anemia .  .  1,712,166  50.0  1.47  1.270 

Secondary  anemia 3,737)i6o  61.0  0.81  0.790 

Chlorosis 3,205,000  34.5  0.55  0.695 

Method. — The  red  cells  are  coimted  and  the  percentage  of 
red  cells  calculated  as  for  the  color  index. 

The  volume  percentage  is  obtained  with  the  hematocrit 
as  follows:  Fill  the  hematocrit  oubes  (Fig.  81)  with  blood,  and 
before  coagulation  takes  place  insert  them  in  the  frame  and 
centrifugalize  for  three  minutes  at  about  8000  to  10,000 


246  THE   BLOOD 

revolutions  a  minute.  The  red  cells  collect  at  the  bottom 
and,  normally,  make  up  one-half  of  the  total  column  of  blood. 
Multiply  the  height  of  the  layer  of  red  cells  (as  indicated  by 
the  graduations  upon  the  side  of  the  tube)  by  2  to  obtain  the 
volume  percentage.  When  the  examination  cannot  be  made 
immediately  after  the  blood  is  obtained,  the  method  of 
Larrabee  is  available.  This  consists  in  mixing  a  trace  of  so- 
dium oxalate  with  a  few  drops  of  blood  to  prevent  coagulation, 
drawing  this  mixture  into  a  heavy-walled  tube  of  about 
2 -mm.  caliber,  closing  the  ends  with  a  rubber  band,  and  wait- 
ing until  sedimentation  is  complete — usually  about  three 
days.     The  height  of  the  column  is  then  measured  with  a 


^   '  ■^-'-'-'-'-'■^■'jj^'-'-r;|;|^i-i-.-^^ 


FiR.  81.— Daland  hematocrit  for  use  with  the  centrifuge. 

millimeter  scale  and  the  percentage  relation  to  the  normal 
calculated. 

After  the  volume  of  the  red  cells  and  the  red  corpuscle 
count  are  thus  expressed  in  percentages,  divide  the  former 
by  the  latter  to  find  the  volume  index.  Example:  Suppose 
the  volume  percentage  is  80  (the  reds  reaching  to  mark  40  on 
hematocrit  tube)  and  that  the  red  count  is  50  per  cent,  of 
the  normal  (2,500,000  per  c.mm.),  then  f^,  or  1.6,  is  the  vol- 
ume index. 

VI.  ENUMERATION  OF  LEUKOCYTES 

The  normal  number  of  leukocytes  varies  from  5000  to 
10,000  per  cubic  millimeter  of  blood.  The  number  is 
larger  in  robust  individuals  than  in  poorly  nourished 


ENUMERATION   OF  LEUKOCYTES  247 

ones,  and,  if  disease  be  excluded,  may  be  taken  as  a 
rough  index  of  the  individual's  nutrition.  Since  it  is 
well  to  have  a  definite  standard,  75CX)  is  generally  adopted 
as  the  normal  for  the  adult.  With  children  the  number 
is  somewhat  greater,  counts  of  12,000  and  15,000  being 
common  in  healthy  children  under  twelve  years  of  age. 

Decrease  in  Number  of  Leukocytes 

Decrease  in  number  of  leukocytes,  or  leukopenia,  is  not 
important.  It  is  common  in  persons  who  are  poorly 
nourished,  although  not  actually  sick.  The  infectious  dis- 
eases in  which  leukocytosis  is  absent  (see  p.  251)  often 
cause  a  slight  decrease  of  leukocytes.  Chlorosis  may 
produce  leukopenia,  as  also  pernicious  anemia,  which 
usually  gives  it  in  contrast  to  the  secondary  anemias, 
which  are  frequently  accompanied  by  leukocytosis. 
Leukocyte  counts  are,  therefore,  of  some  aid  in  the  dif- 
ferential diagnosis  of  these  conditions. 

Increase  in  Number  of  Leukocytes 

Increase  in  number  of  leukocytes  is  common  and  of 
great  importance.  It  may  be  considered  under  two 
heads: 

A.  Increase  of  leukocytes  due  to  chemotaxis  and 
stimulation  of  the  blood-making  organs,  or  leukocytosis. 
The  increase  affects  one  or  more  of  the  normal  varieties. 

B.  Increase  of  leukocytes  due  to  leukemia.  Normal 
varieties  are  increased,  but  the  characteristic  feature  is 
the  appearance  of  great  numbers  of  abnormal  cells. 

The  former  may  be  classed  as  a  transient,  the  latter,  as 
a  permanent,  increase. 


248  THE   BLOOD 

A.  Leukocytosis 

This  term  is  variously  used.  By  some  it  is  applied  to 
any  increase  in  number  of  leukocytes;  by  others  it  is 
restricted  to  increase  of  the  polymorphonuclear  neutro- 
phiHc  variety.  As  has  been  indicated,  it  is  here  taken 
to  mean  a  transient  increase  in  number  of  leukocytes, 
that  is,  one  caused  by  chemotaxis  and  stimulation  of  the 
blood-producing  structures,  in  contrast  to  the  permanent 
increase  caused  by  leukemia. 

By  chemotaxis  is  meant  that  property  of  certain  agents 
by  which  they  attract  or  repel  living  cells — positive 
chemotaxis  and  negative  chemotaxis  respectively.  An 
excellent  illustration  is  the  accumulation  of  leukocytes 
at  the  site  of  inflammation,  owing  to  the  positively 
chemotactic  influence  of  bacteria  and  their  products.  A 
great  many  agents  possess  the  power  of  attracting  leuko- 
cytes into  the  general  circulation.  Among  these  are 
many  bacteria  and  certain  organic  and  inorganic  poisons. 

Chemotaxis  alone  will  not  explain  the  continuance  of 
leukocytosis  for  more  than  a  short  time.  It  is  probable 
that  substances  which  are  postitively  chemotactic  also 
stimulate  the  blood-producing  organs  to  increased  forma- 
tion of  leukocytes;  and  in  at  least  one  form  of  leukocy- 
tosis such  stimulation  apparently  plays  the  chief  part. 

As  will  be  seen  later,  there  are  several  varieties  of  leu- 
kocytes in  normal  blood,  and  most  chemotactic  agents 
attract  only  one  variety,  and  either  repel  or  do  not  in- 
fluence the  others.  It  practically  never  happens  that 
all  are  increased  in  the  same  proportion.  The  most 
satisfactory  classification  of  leukocytoses  is,  therefore, 
based  upon  the  type  of  leukocyte  chiefly  affected. 

Theoretically,  there  should  be  a  subdivision  for  each 


ENUMERATION   OF   LEUKOCYTES  249 

variety  of  leukocyte,  e.  g.,  polymorphonuclear  leuko- 
cytosis, lymphocytic  leukocytosis,  eosinophilic  leuko- 
cytosis, large  mononuclear  leukocytosis,  etc.  Practi- 
cally, however,  only  two  of  these,  polymorphonuclear 
leukocytosis  and  lymphocytic  leukocytosis,  need  be  con- 
sidered under  the  head  of  Leukocytosis.  Increase  in 
number  of  the  other  leukocytes  will  be  considered 
when  the  individual  cells  are  described  (see  pp.  279-291). 
They  are  present  in  the  blood  in  such  small  numbers 
normally  that  even  a  marked  increase  scarcely  affects 
the  total  leukocyte  count;  and,  besides,  substances 
which  attract  them  into  the  circulation  frequently  repel 
the  polymorphonuclears,  so  that  the  total  number  of 
leukocytes  may  actually  be  decreased. 

The  polymorphonuclear  neutrophiles  are  capable  of 
active  ameboid  motion,  and  are  by  far  the  most  numerous 
of  the  leukocytes.  Lymphocytes  are  about  one-third 
as  numerous  and  have  little  independent  motion.  As 
one  would,  therefore,  expect,  marked  differences  exist 
between  the  two  types  of  leukocytosis:  polynuclear 
leukocytosis  is  more  or  less  acute,  coming  on  quickly  and 
often  reaching  high  degree;  whereas  lymphocytic  leuko- 
cytosis is  more  chronic,  comes  on  more  slowly,  and  is 
never  so  marked. 

1.  Polymorphonuclear  Neutrophilic  Leukocytosis. — 
Polymorphonuclear  leukocytosis  may  be  either  physi- 
ologic or  pathologic.  A  count  of  20,000  would  be  con- 
sidered a  marked  leukocytosis;  of  30,000,  high;  above 
50,000,  extremely  high. 

(i)  Physiologic  Polymorphonuclear  Leukocjrtosis. — 
This  is  never  very  marked,  the  count  rarely  exceeding 
15,000  per  cubic  millimeter.    It  occurs:  (a)  In  the  new- 


250  THE    BLOOD 

born;  (b)  in  pregnancy;  (c)  during  digestion,  and  (d) 
after  cold  baths.  There  is  moderate  leukocytosis  in  the 
moribund  state:  this  is  commonly  classed  as  physiologic, 
but  is  probably  due  mainly  to  terminal  infection. 

The  increase  in  these  conditions  is  not  limited  to  the 
polymorphonuclears.  Lymphocytes  are  fikewise  in- 
creased in  varying  degrees,  most  markedly  in  the  new- 
born. 

In  view  of  the  leukocytosis  of  digestion,  the  hour 
at  which  a  leukocyte  count  is  made  should  always  be 
recorded.  Digestive  leukocytosis  is  most  marked  three 
to  five  hours  after  a  hearty  meal  rich  in  protein.  It  is 
absent  in  pregnancy  and  when  leukocytosis  from  any 
other  cause  exists.  It  is  usually  absent  in  cancer  of  the 
stomach,  a  fact  which  may  be  of  some  help  in  the  diag- 
nosis of  this  condition,  but  repeated  examinations  are 
essential. 

(2)  Pathologic  Polymorphonuclear  Leukocytosis. — 
In  general,  the  response  of  the  leukocytes  to  chemotaxis 
is  a  conservative  process.  It  has  been  compared  to  the 
gathering  of  soldiers  to  destroy  an  invader.  This  is 
accomplished  partly  by  means  of  phagocytosis — actual 
ingestion  of  the  enemy — and  partly  by  means  of  chemic 
substances  which  the  leukocytes  produce. 

In  those  diseases  in  which  leukocytosis  is  the  rule  the 
degree  of  leukocytosis  depends  upon  two  factors:  the 
severity  of  the  infection  and  the  resistance  of  the  individual. 
A  well-marked  leukocytosis  usually  indicates  good  resist- 
ance, A  mild  degree  means  that  the  body  is  not  react- 
ing well,  or  else  that  the  infection  is  too  slight  to  call 
forth  much  resistance.  Leukocytosis  may  be  absent 
altogether  when  the  infection  is  extremely  mild,  or  when 


ENUMERATION    OF   LEUKOCYTES  25 1 

it  is  SO  severe  as  to  overwhelm  the  organism  before  it  can 
react.  When  leukocytosis  is  marked,  a  sudden  fall  in 
the  count  may  be  the  first  warning  of  a  fatal  issue. 
These  facts  are  especially  true  of  pneumonia,  diphtheria, 
and  abdominal  inflammations,  in  which  conditions  the 
degree  of  leukocytosis  is  of  considerable  prognostic  value. 
The  classification  here  given  follows  Cabot: 
{a)  Infectious  and  Inflammatory. — The  majority  of 
infectious  diseases  produce  leukocytosis.  The  most  not- 
able exceptions  are  influenza,  malaria,  measles,  tuber- 
culosis, except  when  invading  the  serous  cavities  or 
when  complicated  by  mixed  infection,  and  typhoid  fever, 
in  which  leukocytosis  indicates  an  inflammatory  com- 
plication. 

All  inflammatory  and  suppurative  diseases  cause  leu- 
kocytosis, except  when  slight  or  well  walled  off.  Appen- 
dicitis has  been  studied  with  especial  care  in  this  connec- 
tion, and  the  conclusions  now  generally  accepted  prob- 
ably hold  good  for  most  acute  intra-abdominal  inflam- 
mations. A  marked  leukocytosis  (20,000  or  more) 
nearly  always  indicates  abscess,  peritonitis,  or  gan- 
grene, even  though  the  clinical  signs  be  slight.  Absence 
of  or  mild  leukocytosis  indicates  a  mild  process,  or  else 
an  overwhelmingly  severe  one;  and  operation  may  safely 
be  postponed  unless  the  abdominal  signs  are  very  marked. 
On  the  other  hand,  no  matter  how  low  the  count,  an  in- 
creasing leukocytosis — counts  being  made  hourly — indi- 
cates a  spreading  process  and  demands  operation,  regard- 
less of  other  symptoms. 

.  Leukocyte  counts  alone  are  often  disappointing,  but  are 
of  much  more  value  when  considered  in  connection  with 
a  differential  count  of  polymorphonuclears  (see.  p.  ^28^) . 

cOLLIZCslE  0I--  OtfTtC. 


252  THE   BLOOD 

(b)  Malignant  Disease. — Leukocytosis  occurs  in  about 
one-half  of  the  cases  of  malignant  disease.  In  many 
instances  it  is  probably  independent  of  any  secondary 
infection,  since  it  occurs  in  both  ulcerative  and  non- 
ulcerative cases.  It  seems  to  be  more  common  in  sar- 
coma than  in  carcinoma.  Very  large  counts  are  rarely 
noted. 

(c)  Posthemorrhagic. — Moderate  leukocytosis  follows 
hemorrhage  and  disappears  in  a  few  days. 

(d)  Toxic. — This  is  a  rather  obscure  class,  which  in- 
cludes gout,  chronic  nephritis,  acute  yellow  atrophy 
of  the  Hver,  ptomain-poisoning,  prolonged  chloroform 
narcosis,  and  quinin-poisoning.  Leukocytosis  may  or 
may  not  occur  in  these  conditions,  and  is  not  important. 

(e)  Drugs. — This  also  is  an  unimportant  class.  Most 
tonics  and  stomachics  and  many  other  drugs  produce  a 
slight  leukocytosis. 

2.  Lymphocytic  Leukocytosis. — This  is  characterized 
by  an  increase  in  the  total  leukocyte  count,  accom- 
panied by  an  increase  in  the  percentage  of  lymphocytes. 
The  word  ''lymphocytosis"  is  often  used  in  the  same 
sense.  It  is  better,  however,  to  use  the  latter  as  refer- 
ring to  any  increase  in  the  absolute  number  of  lympho- 
cytes, without  regard  to  the  total  count,  since  an  ab- 
solute increase  in  number  of  lymphocytes  is  frequently 
accompanied  by  a  normal  or  subnormal  leukocyte  count, 
owing  to  loss  of  polymorphonuclears. 

Lymphocytic  leukocytosis  is  probably  due  more  to 
stimulation  of  blood-making  organs  than  to  chemotaxis. 
It  is  less  common,  and  is  rarely  so  marked  as  a  poly- 
morphonuclear leukocytosis.  When  marked,  the  blood 
cannot  be  distinguished  from  that  of  lymphatic  leukemia. 


ENUMERATION   OF  LEUKOCYTES  253 

A  marked  lymphocytic  leukocytosis  occurs  in  pertussis, 
and  is  of  value  in  diagnosis.  It  appears  early  in  the 
catarrhal  stage,  and  persists  until  after  convalescence. 
The  average  leukocyte  count  is  about  17,000,  lympho- 
cytes predominating.  There  is  moderate  lymphocytic 
leukocytosis  in  other  diseases  of  childhood,  as  rickets, 
scurvy,  and  especially  hereditary  syphiUs,  where  the 
blood-picture  may  approach  that  of  pertussis.  It  must 
be  borne  in  mind  in  this  connection  that  lymphocytes 
are  normally  more  abundant  in  the  blood  of  children 
than  in  that  of  adults. 

Slight  lymphocytic  leukocytosis  occurs  in  many  other 
pathologic  conditions,  but  is  of  little  significance. 

B.     Leukemia 

This  is  an  idiopathic  disease  of  the  blood-making 
organs,  which  is  accompanied  by  an  enormous  increase 
in  number  of  leukocytes.  The  leukocyte  count  some- 
times reaches  1,000,000  per  cubic  millimeter,  and  leu- 
kemia is  always  to  be  suspected  when  it  exceeds  50,000. 
Lower  counts  do  not,  however,  exclude  it.  The  subject 
is  more  fully  discussed  later  (see  p.  315). 

Method  of  Counting  Leukocytes 

The  leukocytes  are  counted  with  one  of  the  hemacy- 
tometers already  described  (see  pp.  234-244).  Numer- 
ous modifications  of  the  original  ruling  have  been  intro- 
duced, notably  the  Neubauer,  the  Zappert,  and  the 
Tiirck  (Fig.  82),  which  give  a  ruled  area  of  9  sq.mm., 
the  center  having  the  Thoma  ruling.  They  were  de- 
vised for  counting  the  leukocytes  in  the  same  speci- 
men with  the  red  corpuscles.    The  red  cells  are  counted 


254 


THE   BLOOD 


in  the  central  portion  in  the  usual  manner,  after  which 
all  the  leukocytes  in  the  whole  area  of  9  sq.  mm.  are 
counted;  and  the  number  in  a  cubic  millimeter  of  un- 
diluted blood  is  then  easily  calculated.    Leukocytes  are 


Fig.  82. — Turck  niling  of  counting  chamber. 


easily  distinguished  from  red  cells,  especially  when 
Toisson's  diluting  fluid  is  used.  This  method  may 
be  used  with  the  older  Thoma  ruling  by  adjusting  the 
microscopic  field  to  a  definite  size,  and  counting  a  suffi- 
cient number  of  fields,  as  described  later. 


ENUMERATION    OF   LEUKOCYTES 


255 


Although  less  convenient,  it  is  more  accurate  to  count 
the  leukocytes  separately,  with  less  dilution  of  the  blood, 
as  follows: 


Technic. — A  larger  drop  of  blood  is  required  than  for 
counting  the  erythrocytes,  and  more  care  in  filling  the  pipet. 
Boggs  has  suggested  a  device  (Fig.  83)  which  enables  one  to 


Fig.  83. — Boggs'  "throttle  control"  for  blood-counting'pipet,  and  enlarged  diagram  show- 
ing construction  of  the  throttle. 

draw  in  the  blood  more  slowly  and  hence  more  accurately. 
He  cuts  the  rubber  tube  and  inserts  a  Wright  "throttle." 
This  consists  of  a  section  of  glass  tubing  in  which  a  capillary 
tube  drawn  out  to  a  fine  thread  is  cemented  with  sealing  wax. 
After  sealing  in  place  the  tip  is  broken  off  with  forceps,  so 
that  upon  gentle  suction  it  will  just  allow  air  to  pass. 

Use  the  pipet  with  11  engraved  above  the  bulb.    Suck  the 
blood  to  the  mark  0.5  or  i.o,  and  the  diluting  fluid  to  the 


256  THE   BLOOD 

mark  11.  This  gives  a  dilution  of  i :  20  or  i :  10,  respectively. 
The  dilution  of  i  :  20  is  easier  to  make.  Mix  well  by  shaking 
in  all  directions  except  in  the  long  axis  of  the  pipet;  blow  out 
2  or  3  drops,  place  a  drop  in  the  counting  chamber,  and 
adjust  the  cover  as  already  described  (see  p.  237). 

Examine  with  a  low  power  to  see  that  the  cells  are  evenly 
distributed.  Count  with  the  i6-mm.  objective  and  a  high 
eye-piece,  or  with  the  long-focus  4  mm.  and  a  low  eye-piece. 
An  8-mm.  objective  will  be  found  very  satisfactory  for  this 
purpose.  As  one  gains  experience  one  will  rely  more  upon 
the  lower  powers. 

With  the  Thoma  ruling  count  all  the  leukocytes  in  the 
square  millimeter,  multiply  by  10  to  find  the  number  in 
I  c.mm.  of  diluted  blood,  and  by  the  dilution  to  find  the 
number  per  cubic  millimeter  of  undiluted  blood.  In  every 
case  at  least  200  leukocytes  must  be  counted  as  a  basis  for 
calculation,  and  it  is  much  better  to  count  500.  This  will 
necessitate  examination  of  several  drops  from  the  pipet. 
With  the  Zappert  and  Tiirck  rulings  a  sufficient  number  can 
usually  be  counted  in  one  drop,  but  the  opportunity  for  error 
is  ver\'  much  greater  when  only  one  drop  is  examined. 

In  routine  work  the  author's  modification  of  the  "circle" 
method  is  ver\'  satisfactory.  It  requires  a  4-mm.  objective, 
and  is,  therefore,  especially  desirable  for  beginners,  who  are 
usually  unable  accurateh'  to  identify  leukoc\'tes  with  a  lower 
power.  The  student  is  frequently  confused  by  particles  of 
dirt,  remains  of  red  cells,  and  yeast  cells  which  frequently 
grow  in  the  diluting  fluid.  Draw  out  the  sliding  tube  of  the 
microscope  until  the  field  of  vision  is  such  as  shown  in  Fig. 
84.  One  side  of  the  field  is  tangent  to  one  of  the  ruled  Imes, 
A,  while  the  opposite  side  just  cuts  the  comers,  B  and  C,  of 
the  seventh  squares  in  the  rows  above  and  below  the  diameter 
line.  When  once  adjusted,  a  scratch  is  made  upon  the  draw- 
tube,  so  that  for  future  counts  the  tube  has  only  to  be  drawn 
out  to  the  mark.    The  area  of  this  microscopic  field  is  one- 


ENUMERATION   OF   LEUKOCYTES 


257 


tenth  of  a  square  millimeter.  With  a  dilution  of  r  :  20,  count 
the  leukocytes  in  20  such  fields  upon  different  parts  of  the  disk 
without  regard  to  the  ruled  lines,  and  to  their  sum  add  two 
ciphers.  With  dilution  of  i  :  10,  coimt  10  such  fields,  and 
add  two  ciphers.    Thus,  with  i  :  10  dilution,  if  150  leukocytes 


--f r 

1           1 

'     I      ' 
1     '      1 

1                 1 

r 

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r 

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--T ^---_4-----l-  .r-+---i 

,              .              1              III 

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L  -  4.     -     J.  - 

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Fig.  84. — Size  of  field  required  in  counting  leukocytes  as  described  in  the  text. 


were  counted  in  10  fields,  the  leukocyte  count  would  be  15,000 
per  cubic  millimeter.  To  compensate  for  possible  unevenness 
of  distribution,  it  is  best  to  count  a  row  of  fields  horizontally 
and  a  row  vertically  across  the  disk.  This  method  is  applica- 
ble to  any  degree  of  dilution  of  the  blood,  and  is  simple  to 
17 


258  THE   BLOOD 

remember:  •one  always  counts  a  number  of  fields  equal  to  the 
number  of  times  the  blood  has  been  diluted,  and  adds  two  ciphers. 
E\'idence  of  the  convenience  of  using  a  circle  of  this  size  is 
afforded  by  its  adoption  in  the  new  Thoma-Metz  instrument. 

It  is  sometimes  impossible  to  obtain  the  proper  size  of 
field  with  the  objectives  and  eye-pieces  at  hand.  In  such  case 
place  a  cardboard  disk  with  a  circular  opening  upon  the  dia- 
phragm of  the  eye-piece,  and  adjust  the  size  of  the  field  by 
drawing  out  the  tube.  The  circular  opening  can  be  cut  with 
a  cork-borer. 

Diluting  Fluids. — The  diluting  fluid  should  dissolve  the 
red  corpuscles  so  that  they  will  not  obscure  the  leukocytes. 
The  simplest  fluid  is  a  0.5  per  cent,  solution  of  acetic  acid. 
More  satisfactory  is  the  following:  glacial  acetic  acid,  i  c.c. ; 
I  per  cent,  aqueous  solution  of  gentian-violet,  i  c.c. ;  distilled 
water,  100  c.c.  These  solutions  must  be  filtered  frequently 
to  remove  yeasts  and  molds. 

Vn.  ENUMERATION  OF  BLOOD-PLAQUES 

The  average  normal  number  of  plaques  is  variously 
given  as  200,000  to  700,000  per  cubic  millimeter  of 
blood.  Many  of  the  counts  were  obtained  by  workers 
who  used  unreliable  methods.  Using  their  new  method, 
Wright  and  Kinnicutt  found  the  normal  average  to  range 
from  263,000  to  336,000.  Physiologic  variations  are 
marked;  thus,  the  number  increases  as  one  ascends  to  a 
higher  altitude,  and  is  higher  in  winter  than  in  summer. 
There  are  unexplained  variations  from  day  to  day; 
hence  a  single  abnormal  count  should  not  be  taken  to 
indicate  a  pathologic  condition. 

Pathologic  variations  are  often  very  great.  Owing  to 
lack  of  knowledge  as  to  the  origin  of  the  platelets  and  to 
the  earlier  imperfect  methods  of  counting,  the  clinical 


ENUMERATION    OF    BLOOD-PLAQUES  259 

significance  of  these  variations  is  uncertain.     The  fol- 
lowing facts  seem,  however,  to  be  established : 

(a)  In  acute  infectious  diseases  the  number  is  sub- 
normal or  normal.  If  the  fever  ends  by  crisis,  the  crisis 
is  accompanied  by  a  rapid  and  striking  increase. 

(b)  In  secondary  anemia  plaques  are  generally  in- 
creased, although  sometimes  decreased.  In  pernicious 
anemia  they  are  always  greatly  diminished,  and  an 
increase  should  exclude  the  diagnosis  of  this  disease. 

(c)  They  are  decreased  in  chronic  lymphatic  leukemia, 
and  greatly  increased  in  the  myelogenous  form. 

(d)  In  purpura  haemorrhagica  the  number  is  enor- 
mously diminished. 

Blood-plaques  are  difficult  to  count,  owing  to  the 
rapidity  with  which  they  disintegrate,  and  to  their  great 
tendency  to  adhere  to  any  foreign  body  and  to  each 
other. 

Method  of  Kemp,  Calhoun,  and  Harris. — Wash  the  finger 
well  and  allow  a  few  minutes  to  elapse  for  the  circulation 
to  become  normal.  Prick  the  finger  lightly  with  a  blood- 
lancet,  regulating  the  depth  of  the  puncture  so  that  the  blood 
will  not  flow  without  gentle  pressure.  Quickly  dip  a  clean 
glass  rod  into  a  vessel  containing  diluting  and  fixing  fluid, 
and  place  two  or  three  good-sized  drops  upon  the  finger  over 
the  puncture.  Then  exert  gentle  pressure  above  the  puncture 
so  that  a  small  drop  of  blood  will  exude  mto  the  fluid.  Mix 
the  two  by  passing  the  rod  lightly  several  times  over  the  sur- 
face of  the  blended  drop.  (Some  workers  first  place  a  drop 
of  the  fluid  upon  the  finger  and  then  make  the  puncture 
through  it,  this  necessitating  less  care  as  to  depth  of  the 
puncture.)  Now  transfer  a  drop  of  the  diluted  blood  from 
the   finger  to  a  watch-glass   which  contains  2  or  3  drops 


26o  THE   BLOOD 

of  the  fluid,  and  mix  well.  From  this,  transfer  a  drop  to  the 
counting  slide  of  the  hemacytometer,  and  cover.  An  ordi- 
nary thin  cover  will  answer  for  this  purpose,  and  is  preferable 
because  it  allows  the  use  of  a  higher  power  objective.  Allow 
the  slide  to  stand  for  at  least  five  minutes,  and  then  with 
a  4-mm.  or  higher  objective  count  the  plaques  and  the  red 
corpuscles  in  a  definite  number  of  squares.  At  least  loo 
plaques  must  be  counted.  The  number  of  red  corpuscles 
per  cubic  millimeter  of  blood  having  been  previously  ascer- 
tained in  the  usual  manner  (see  p.  234),  the  number  of 
plaques  can  easily  be  calculated  by  the  following  equation: 

r:  p::R:P;  and  P  =  ^Jlj?. 
r 

r  represents  the  number  of  red  corpuscles  in  any  given  num- 
ber of  squares;  p,  the  number  of  plaques  in  the  same  squares; 
R,  the  total  number  of  red  corpuscles  per  cubic  millimeter 
of  blood;  and  P,  the  number  of  plaques  per  cubic  milH- 
meter. 

Beginners  are  apt  to  take  too  much  blood  and  not  to  dilute 
it  enough.  Best  results  are  attained  when  there  are  only  one 
or  two  plaques  in  a  small  square.  With  insufficient  dilution, 
the  platelets  are  more  or  less  obscured  by  the  red  cells. 

The  following  diluting  and  fixing  fluid  is  recommended: 

Formalin 10  c.c; 

Aqueous  solution  sodium  chlorid  (i  per  cent.) ....  150  c.c. 
(Color  with  methyl-violet  if  desired.) 

This  fluid  is  cheap  and  easily  prepared,  and  keeps  indefi- 
nitely. It  fixes  the  plaques  quickly  without  clumping,  and 
does  not  clump  nor  decolorize  the  reds.  It  has  a  low  specific 
gravity,  and  hence  allows  the  plaques  to  settle  upon  the  ruled 
area  along  with  the  reds.  Fluids  of  high  specific  gravity 
cause  the  plaques  to  float  so  that  they  do  not  appear  in  the 
same  plane  with  the  reds  and  the  ruled  lines. 


STUDY  OF  STAINED  BLOOD  26 1 

Method  of  Wright  and  Eonnicutt. — ^This  new  method  is 
simple,  appears  to  be  accurate,  and  certainly  yields  uniform 
results. 

The  plaques  are  counted  with  the  hemacytometer  already 
described,  using  a  dilution  of  i  :  100.  The  diluting  fluid  con- 
sists of  2  parts  of  an  aqueous  solution  of  "brilliant  cresyl 
blue"  (i  :  300)  and  3  parts  of  an  aqueous  solution  of  po- 
tassium cyanid  (i  :  1400).  These  two  solutions  must  be 
kept  in  separate  bottles  and  mixed  and  filtered  immediately 
before  using.  After  the  blood  is  placed  in  the  comi ting- 
chamber  it  is  allowed  to  stand  for  ten  minutes  or  longer  in 
order  that  the  plaques  may  settle.  The  plaques  appear  as 
rounded,  lilac-colored  bodies;  the  reds  are  decolorized,  ap- 
pearing only  as  shadows. 

The  leukocytes  are  stained  and  may  be  counted  at  the 
same  time. 

Vm.  STUDY  OF  STAINED  BLOOD 
A.   Making  and  Staining  Blood-films 

1 .  Spreading  the  Film. — Thin,  even  films  are  essential 
to  accurate  and  pleasant  work.  They  more  than  com- 
pensate for  the  time  spent  in  learning  to  make  them. 
There  are  certain  requisites  for  success  with  any  method : 
(a)  The  slides  and  covers  must  be  perfectly  clean: 
thorough  washing  with  soap  and  water  and  rubbing 
with  alcohol  will  usually  suffice;  (b)  the  drop  of  blood 
must  not  be  too  large;  (c)  the  work  must  be  done  quickly, 
before  coagulation  begins. 

The  blood  is  obtained  from  the  finger-tip  or  the  lobe 
of  the  ear,  as  for  a  blood  count;  only  a  very  small  drop  is 
required,  usually  about  the  size  of  a  pin-head. 

Ehrlich's  Two  Cover-glass  Method. — ^This  method  is  very 
widely  used,  but  considerable  practice  is  required  to  get  good 


262 


THE   BLOOD 


results.  Touch  a  cover-glass  to  the  top  of  a  small  drop  of 
blood,  and  place  it,  blood  side  down,  upon  another  cover- 
glass.  If  the  drop  be  not  too  large,  and  the  covers  be  per- 
fectly clean,  the  blood  will  spread  out  in  a  very  thin  layer. 
Just  as  it  stops  spreading,  before  it  begins  to  coagulate,  pull 
the  covers  quickly  but  firmly  apart  on  a  plane  parallel  to  their 
surfaces  (Fig.  85).  It  is  best  to  handle  the  covers  with  for- 
ceps, since  the  moisture  of  the  fingers  may  produce  artifacts. 
The  forceps  must  have  a  firm  grasp. 


Fig  85. — Spreading  the  film:  two  cover-glass  method. 

This  method  is  especially  to  be  recommended  for  very 
accurate  differential  counts,  since  all  the  leukocytes  in 
the  drop  will  be  found  on  the  two  covers.  One  of  the 
covers  is  usually  much  better  spread  than  the  other. 


Two-slide  Method. — Take  a  small  drop  of  blood  upon  a 
clean  slide  about  ^  inch  from  the  end,  using  care  that  the 
slide  does  not  touch  the  skin.  Place  the  end  of  a  second  slide 
against  the  surface  of  the  first  at  an  angle  of  45  degrees,  and 
draw  it  up  against  the  drop  of  lilood,  which  will  immediately 
run  across  the  end,  filling  the  angle  between  the  two  sHdes. 
Now  push  the  "spreader  slide"  back  along  the  other  in  the 
manner  indicated  in  Fig.  86.     The  blood  will  follow.      The 


STUDY   OF   STAINED   BLOOD 


263 


thickness  of  the  smear  can  be  regulated  by  changing  the 
angle. 


Fig.  86. — Spreading  the  film:  two-slide  method. 


It  is  very  easy  to  make  large,  thin,  even  films  by  this 
method. 


Fig.  87. — Spreading  the  film:  cigarette-paper  method  applied  to  cover-glasses. 

Cigarette-paper  Method.— This  gives  excellent  results  in 
the  hands  of  the  inexperienced  if  directions  are  carefully  fol-. 
lowed,  but  its  only  advantage  over  the  two-slide  method  is 


264  THE   BLOOD 

that  it  may  be  used  with  covers  as  well  as  with  slides.  A 
very  thin  paper,  such  as  the  "Zig-zag"  brand,  is  best.  Ordi- 
nary cigarette  paper  and  thin  tissue-paper  will  answer,  but 
do  not  give  nearly  so  good  results. 

Cut  the  paper  into  strips  about  f  inch  wide,  across  the  ribs. 
Pick  up  one  of  the  strips  by  the  gummed  edge,  and  touch  its 
opposite  end  to  the  drop  of  blood.  Quickly  place  the  end 
which  has  the  blood  against  a  slide  or  a  large  cover-glass  held 
in  a  forceps.  The  blood  will  spread  along  the  edge  of  the 
paper.  Now  draw  the  paper  evenly  across  the  slide  or  cover. 
A  thin  film  of  blood  will  be  left  behind  (Fig.  87). 

The  films  may  be  allowed  to  dry  in  the  air,  or  may  be 
dried  by  gently  heating  high  above  a  flame  (where  one 
can  comfortably  hold  the  hand).  Such  films  will  keep 
for  years,  but  for  some  stains  they  must  not  be  more 
than  a  few  weeks  old.  They  must  be  kept  away  from 
flies — a  fly  can  work  havoc  with  a  film  in  a  few  minutes. 

When  slides  are  used  the  label  can  be  written  with  a 
soft  lead  pencil  directly  on  the  blood-film,  as  has  been 
suggested  by  von  Ezdorf. 

2.  Fixing  the  Film. — In  general,  films  must  be  "fixed" 
before  they  are  stained.  Fixation  may  be  accomplished 
by  chemicals  or  by  heat.  Those  stains  which  are  dis- 
solved in  methyl  alcohol  combine  fixation  with  the  staining 
process. 

Chemic  Fixation. — Soak  the  film  one  to  five  minutes  in 
pure  methyl  alcohol  or  absolute  ethyl  alcohol,  or  one-half 
hour  or  longer  in  equal  parts  of  absolute  alcohol  and  ether. 
One  minute  in  i  per  cent,  formalin  in  alcohol  is  preferred 
by  some,  especially  for  the  carbol-thionin  stain.  Chemic 
fixation  may  precede  eosin-methylene-blue  and  other  simple 
stains. 


STUDY  OF  STAINED  BLOOD  265 

Heat  Fixation. — ^This  may  precede  any  of  the  methods 
which  do  not  combine  fixation  with  the  staining  process;  it 
must  be  used  with  EhrHch's  triple  stain.  The  best  method  is 
to  place  the  film  in  an  oven,  raise  the  temperature  to  150°  C, 
and  allow  to  cool  slowly.  Without  an  oven,  the  proper  degree 
of  fixation  is  difficult  to  attain.  Kowarsky  has  devised  a 
small  plate  of  two  layers  of  copper  (Fig.  88),  upon  which 
the  films  are  placed  together  with  a  crystal  of  urea.  The 
plate  is  heated  over  a  flame  until  the  urea  melts,  and  is  then 
set  aside  to  cool.  This  is  said  to  give  the  proper  degree  of 
fixation,  but  in  the  writer's  experience  the  films  have  always 
been  imderheated.    He  obtains  better  results  by  use  of  tar- 


Fig  88. — Kowarsky's  plate  for  fixing  blood  (Ellopstock  and  Kowarsky). 

taric  acid  crystals  (melting-point,  i68°-i7o°  C).  The  plate, 
upon  which  have  been  placed  the  cover-glasses,  film  side 
down,  and  a  crystal  of  the  acid,  is  heated  over  a  low  flame 
until  the  crystal  has  completely  melted.  It  should  be  held 
sufficiently  high  above  the  flame  that  the  heating  will  require 
five  to  seven  minutes.  The  covers  are  then  removed.  Freshly 
made  films  of  normal  blood  should  be  allowed  to  remain  upon 
the  plate  for  a  minute  or  two  after  heating  has  ceased.  Fresh 
films  require  more  heat  than  old  ones,  and  normal  blood 
more  than  the  blood  of  pernicious  anemia  and  leukemia. 

Blood  films  can  be  satisfactorily  fixed  for  most  purposes 
by  covering  with  absolute  alcohol,  quickly  dashing  off  the 
excess,  and  igniting  the  remainder. 


266  THE    BLOOD 

3.  Staining  the  Film. — The  anilin  dyes,  which  are 
extensively  used  in  blood  work,  are  of  two  general  classes : 
basic  dyes,  of  which  methylene-blue  is  the  type;  and  acid 
dyes,  of  which  eosin  is  the  type.  Nuclei  and  certain 
other  structures  in  the  blood  are  stained  by  the  basic 
dyes,  and  are  hence  called  basophilic.  Certain  struc- 
tures take  up  only  acid  dyes,  and  are  called  acidophilic, 
oxyphilic,  or  eosinophilic.  Certain  other  structures  are 
stained  only  by  combinations  of  the  two,  and  are  called 
neutrophilic.  Recognition  of  these  staining  properties 
marked  the  beginning  of  modern  hematology. 

(i)  Eosin  and  Methylene-blue. — In  many  instances 
this  stain  will  give  all  the  information  desired.  It  is 
especially  useful  in  studying  the  red  corpuscles.  Nuclei, 
basophilic  granules,  and  all  blood  parasites  are  blue; 
erythrocytes  are  red  or  pink ;  eosinophilic  granules,  bright 
red.  Neutrophilic  granules  and  blood-plaques  are  not 
stained. 

(i)  Fix  the  film  by  heat  or  chemicals. 

(2)  Stain  about  five  minutes  with  a  0.5  per  cent,  alcoholic 
solution  of  eosin,  diluted  one-half  with  water. 

(3)  Rinse  in  water. 

(4)  Stain  one-half  to  one  minute  with  half-saturated  aque- 
ous solution  of  methylene-blue. 

(5)  Rinse  well,  dry,  and  mount.  Films  upon  slides  may 
be  examined  with  an  oil-immersion  objective  without  a  cover- 
glass. 

(2)  Ehrlich's  Triple  Stain.— This  was  the  standard 
blood-stain  for  many  years,  but  is  now  little  used.  It 
is  probably  the  best  for  neutrophilic  granules.  It  is 
difficult  to  make,  and  should  be  purchased  ready  pre- 


STUDY  OF  STAINED  BLOOD  267 

pared  from  a  reliable  dealer.  Nuclei  are  stained  pale 
blue  or  greenish  blue;  erythrocytes,  orange;  neutro- 
philic granules,  violet;  and  eosinophilic  granules,  copper 
red.  Basophilic  granules  and  blood-plaques  are  not 
stained. 

Success  in  staining  depends  largely  upon  proper  fixa- 
tion. The  film  must  be  carefully  fixed  by  heat:  under- 
heating  causes  the  erythrocytes  to  stain  red;  overheat- 
ing, pale  yellow.  The  staining  fluid  is  applied  for  five 
to  fifteen  minutes,  and  the  preparation  is  rinsed  quickly 
in  water,  dried,  and  mounted.  Subsequent  application 
of  Loffler's  methylene-blue  for  one-half  to  one  second  will 
bring  out  the  basophilic  granules  and  improve  the 
nuclear  staining,  but  there  is  considerable  danger  of 
overstaining. 

(3)  Polychrome  Methylene-blue-eosin  Stains. — These 
stains,  mostly  outgrowths  of  the  original  Romanowsky 
method,  have  largely  displaced  other  blood-stains  for  clin- 
ical purposes.  They  stain  differentially  every  normal  and 
abnormal  structure  in  the  blood.  Most  of  them  are 
dissolved  in  methyl  alcohol  and  combine  the  fixing  with 
the  staining  process.  Numerous  methods  of  preparing 
and  applying  these  stains  have  been  devised.  Three 
only  need  be  given  here:  Wright's  stain.  Harlow's  stain, 
and  Jenner's  stain. 

Wright's  Stain. — This  is  one  of  the  best  and  is  the 
most  widely  used  in  this  country.  The  practitioner 
will  find  it  best  to  purchase  the  stain  ready  prepared  or 
to  purchase  the  powder  and  dissolve  it  in  methyl  alcohol 
as  needed.  Most  microscopic  supply-houses  carry  it  in 
stock.  Wright's  most  recent  directions  for  its  prepara- 
tion and  use  are  as  follows: 


268  THE   BLOOD 

Preparation. — To  a  0.5  per  cent,  aqueous  solution  of 
sodium  bicarbonate  add  methylene-blue  (B.  X.  or  "medicin- 
ally pure")  in  the  proportion  of  i  gm.  of  the  dye  to  each  100 
c.c.  of  the  solution.  Heat  the  mixture  in  a  steam  sterilizer 
at  100°  C.  for  one  full  hour,  counting  the  time  after  the  ster- 
ilizer has  become  thoroughly  heated.  The  mixture  is  to  be 
contained  in  a  flask,  or  flasks,  of  such  size  and  shape  that  it 
forms  a  layer  not  more  than  6  cm.  deep.  After  heating, 
allow  the  mixture  to  cool,  placing  the  flask  in  cold  water, 
if  desired,  and  then  filter  it  to  remove  the  precipitate  which 
has  formed  in  it.  It  should,  when  cold,  have  a  deep  purple- 
red  color  when  viewed  in  a  thin  layer  by  transmitted  yellow- 
ish artificial  light.  It  does  not  show  this  color  while  it  is 
warm. 

To  each  100  c.c.  of  the  filtered  mixture  add  500  c.c.  of  a 
0.1  per  cent,  aqueous  solution  of  "yellowish  water-soluble" 
eosin  and  mix  thoroughly.  Collect  the  abundant  precipi- 
tate which  immediately  appears  on  a  filter.  When  the  pre- 
cipitate is  dry,  dissolve  it  in  methylic  alcohol  (Merck's 
"reagent")  in  the  proportion  of  o.i  gm.  to  60  c.c.  of  the  alco- 
hol. In  order  to  facilitate  solution,  the  precipitate  is  to  be 
rubbed  up  with  the  alcohol  in  a  porcelain  dish  or  mortar  with 
a  spatula  or  pestle.  This  alcoholic  solution  of  the  precipitate 
is  the  staining  fluid. 

Application. — i.  Cover  the  film  with  a  noted  quantity  of 
the  staining  fluid  by  means  of  a  medicine-dropper.  There 
must  be  plenty  of  stain  in  order  to  avoid  too  great  evaporation 
and  consequent  precipitation.  When  slides  are  used,  the 
stain  may  be  confined  to  the  smeared  area  by  two  heavy 
grease  pencil  marks. 

2.  After  one  minute  add  to  the  staining  fluid  on  the  fihn 
the  same  quantity  of  distilled  water  by  means  of  a  medicine- 
dropper  and  allow  the  mixture  to  remain  for  two  or  three 
minutes,  according  to  the  intensity  of  the  staining  desired. 
A   longer  period  of   staining  may  produce  a  precipitate. 


STUDY    OF   STAINED   BLOOD  269 

Eosinophilic  granules  are  best  brought  out  by  a  short  period 
of  staining. 

The  quantity  of  the  diluted  fluid  on  the  preparation  should 
not  be  so  large  that  some  of  it  runs  off. 

3.  Wash  the  preparation  in  water  for  thirty  seconds  or 
until  the  thinner  portions  of  the  film  become  yellow  or 
pink  in  color. 

4.  Dry  and  mount  in  balsam. 

The  stain  is  more  conveniently  applied  upon  cover- 
glasses  than  upon  slides.  Films  much  more  than  a  month 
old  do  not  stain  well.  In  some  localities  ordinary  tap- 
water  will  answer  both  for  diluting  the  stain  and  for 
washing  the  film;  in  others,  distilled  water  must  be  used. 
The  dif&culty  here  is  probably  that  the  tap-water  is 
acid  in  reaction.  This  causes  the  nuclei  to  stain  too 
palely.  Other  causes  of  pale  nuclei  are  addition  of  too 
much  or  too  Httle  water  and  the  development  of  formic 
acid  from  the  methyl  alcohol  of  the  staining  fluid. 

When  properly  applied,  Wright's  stain  gives  the  fol- 
lowing picture  (see  Frontispiece) :  erythrocytes,  yellow  or 
pink;  nuclei,  various  shades  of  bluish  purple;  neutrophilic 
granules,  reddish  lilac;  eosinophilic  granules,  bright  red; 
basophilic  granules  of  leukocytes  and  degenerated  red 
corpuscles,  very  dark  bluish  purple;  blood-plaques, 
dark  lilac;  bacteria,  blue.  The  cytoplasm  of  lymphocytes 
is  generally  robin's-egg  blue;  that  of  the  large  mononu- 
clears may  have  a  faint  bluish  tinge.  Malarial  parasites 
stain  characteristically:  the  cytoplasm,  sky-blue;  the 
chromatin,  reddish  purple. 

Harlow's  Stain.— Probably  the  simplest  of  the  poly- 
chrome methylene-blue-eosin  stains,  both  in  preparation 
and  method  of  use,  is  that  devised  by  W.  P.  Harlow,  of 


270  THE   BLOOD 

the  University  of  Colorado.  It  differentiates  granules 
particularly  well,  but  is  not  so  satisfactory  for  demon- 
strating slight  grades  of  polychromatophilia,  because  it 
usually  gives  all  the  red  cells  a  faint  bluish  tinge. 

Preparation. — The  stain  consists  of  two  solutions  used 
separately: 

No.  I.     Eosin,  yellowish,  water  soluble  (Griibler) i  gm.; 

Methyl  alcohol  (Merck's  reagent) 100  c.c; 

No.  2.     Mcthylene-blue    ("  B.   X."    or    Ehrlich's    rectified) 

(Griibler) i  gm. ; 

Mcthj'l  alcohol  (jMerck's  reagent) 100  c.c. 

Application. — (i)  Stain  the  film  without  previous  fixation 
for  one  minute  with  the  eosin  solution. 

(2)  Shake  off  the  excess,  allowing  a  very  little  to  remain, 
and  at  once  cover  with  the  methylene-blue  solution  for  one 
or  two  minutes. 

(3)  Rinse  quickly  in  distilled  water,  dry,  and  mount. 

It  is  well  known  that  pathologic  bloods  will  sometimes 
not  stain  well  with  fluids  which  are  satisfactory  for 
normal  bloods.  Doctors  Peebles  and  Harlow  have  shown 
that  the  various  polychrome  methylene-blue-eosin  stains 
can  be  modified  to  suit  any  blood  by  adding  a  little 
alkali  or  acid.  The  alkali  used  is  a  weak  solution  of 
"potassium  hydrate  by  alcohol"  in  methyl  alcohol;  the 
acid,  glacial  acetic  in  methyl  alcohol.  In  the  case  of  the 
Harlow  stain  it  is  added  to  the  methylene-blue  solution 
only.  The  alkali  solution  also  serves  to  "correct"  old 
fluids  which,  by  reason  of  development  of  formic  acid 
in  the  methyl  alcohol,  do  not  stain  sufficiently  with  the 
blue.  In  general  a  stain  is  satisfactory  when  both  nuclei 
and  neutrophilic  granules  are  clearly  defined. 


STUDY    OF   STAINED    BLOOD  27 1 

Jenner's  Stain. — This  brings  out  leukocytic  granules 
well,  and  is,  therefore,  especially  useful  for  differential 
counting.  It  stains  nuclei  poorly  and  is  much  inferior 
to  Wright's  stain  for  the  malarial  parasite. 

It  may  be  purchased  in  solution,  in  the  form  of  tablets, 
or  as  a  powder,  0.5  gm.  of  which  is  dissolved  in  100  c.c. 
neutral  absolute  methyl  alcohol.  The  blood-film  is 
flooded  with  the  stain  and  after  three  to  five  minutes  is 
rinsed  with  water,  dried  in  the  air,  and  mounted. 

(4)  Carbol-thionin  is  especially  useful  for  the  study 
of  basophilic  granular  degeneration  of  the  red  cells. 
The  method  is  described  on  p.  467.  Nuclei,  malarial 
parasites,  and  basophilic  granules  are  brought  out 
sharply.  Polychromatophilia  is  also  evident.  Fixation 
may  be  by  alcohol-formalin  (see  page  264)  or  saturated 
solution  of  mercuric  chlorid. 

(5)  Pappenheim's  pyronin-methyl  green  (see  p.  467) 
can  be  used  as  a  blood-stain  and  is  very  satisfactory  for 
study  of  the  red  cells,  and  especially  of  the  lymphocytes. 
All  nuclei  are  greenish  blue;  basophilic  granules,  red; 
cytoplasm  of  lymphocytes,  red.  Polychromatophilia  is 
well  demonstrated,  the  affected  cells  taking  more  or  less 
of  the  red  color.    Heat  fixation  is  probably  best. 

B.   Study  of  Stained  Films 

Much  can  be  learned  from  stained  blood-films.  They 
furnish  the  best  means  of  studying  the  morphology  of  the 
blood  and  blood  parasites,  and,  to  the  experienced,  they 
give  a  fair  idea  of  the  amount  of  hemoglobin  and  the 
number  of  red  and  white  corpuscles.  An  oil-immersion 
objective  is  required. 


272  THE   BLOOD 

1.  Erythrocytes. — Normally,  the  red  corpuscles  are 
acidophilic.  The  colors  which  they  take  with  different 
stains  have  been  described.  When  not  crowded  to- 
gether, they  appear  as  circular,  homogeneous  disks,  of 
nearly  uniform  size,  averaging  7.5  /t^  in  diameter  (see  Fig. 
105).  The  center  of  each  is  somewhat  paler  than  the 
periphery.  The  degree  of  pallor  furnishes  a  rough  index 
to  the  amount  of  hemoglobin  in  the  corpuscle.  As 
hemoglobin  is  diminished,  the  central  pale  area  becomes 
larger  and  paler,  producing  the  so-called  "pessary 
forms"  in  which  only  the  periphery  of  the  cell  is  apparent. 
These  forms  indicate  a  low  color  index  and  are  most 
abundant  in  chlorosis.  Red  cells  are  apt  to  be  crenated 
when  the  film  has  dried  too  slowly. 

Pathologically,  red  corpuscles  vary  in  size  and  shape, 
staining  properties,  and  structure. 

(i)  Variations  in  Size  and  Shape  (See  Plate  IX  and 
Fig.  105). — The  cells  may  be  abnormally  small  (called 
microcytes,  5  u.  or  less  in  diameter);  abnormally  large 
{macrocytes,  10  to  12  /m);  or  extremely  large  {megalocytes, 
12  to  25  n).  Abnormal  variation  in  size  is  called  ani- 
socytosis. 

Variation  in  shape  is  often  very  marked.  Oval,  pyri- 
form,  caudate,  saddle-shaped,  and  club-shaped  corpus- 
cles are  common  (Fig.  89).  They  are  called  poikilocytes, 
and  their  presence  is  spoken  of  as  poikilocytosis. 

Red  corpuscles  which  vary  from  the  normal  in  size  and 
shape  are  present  in  most  symptomatic  anemias,  and  in 
the  severer  grades  are  often  very  numerous.  Irregular- 
ities are  particularly  conspicuous  in  leukemia  and  pernic- 
ious anemia,  where,  in  some  instances,  a  normal  erythro- 
cyte is  the  exception.    In  pernicious  anemia  there  is  a 


STUDY  OF  STAINED  BLOOD 


273 


decided  tendency  to  large  size  and  oval  forms,  and  mega- 
locytes  are  rarely  found  in  any  other  condition. 

(2)  Variations  in  Staining  Properties  (See  Plate 
IX). — These  include  polychromatophilia,  basophilic  de- 
generation, and  malarial  stippling.  With  exception  of 
polychromatophilia  they  are  probably  degenerative 
changes. 


A  B  C 

Fig.  89. — Abnormal  red  corpuscles:  A,  Poikilocytosis;  B,  basophilic  granular  degenera- 
tion; C,  malarial  stippling,  the  cell  also  containing  a  tertian  parasite  (X  1000)  (courtesy  of 
Dr.  W.  P.  Harlow). 


(a)  Polychromatophilia. — Some  of  the  corpuscles  par- 
tially lose  their  normal  affinity  for  acid  stains,  and  take 
the  basic  stain  to  greater  or  less  degree.  Wright's  stain 
gives  such  cells  a  faint  bluish  tinge  when  the  condition  is 
mild,  and  a  rather  deep  blue  when  severe.  Sometimes 
only  part  of  a  cell  is  affected.  A  few  polychromatophilic 
corpuscles  can  be  found  in  marked  symptomatic  anemias. 
They  occur  most  abundantly  in  malaria,  leukemia,  and 
pernicious  anemia. 

18 


274  THE    BLOOD 

Polychromatophilia  has  been  variously  interpreted. 
It  is  thought  by  many  to  be  evidence  of  youth  in  a  cell, 
and  hence  to  indicate  an  attempt  at  blood  regeneration. 
There  are  probably  several  forms  referable  to  different 
causes. 

(b)  Basophilic  Granular  Degeneration  {Degeneration  of 
Grawitz). — This  is  characterized  by  the  presence,  within 
the  corpuscle,  of  basophilic  granules  which  vary  in  size 
from  scarcely  visible  points  to  granules  as  large  as  those 
of  basophilic  leukocytes  (Fig.  89.  B) .  The  number  present 
in  a  red  cell  commonly  varies  in  inverse  ratio  to  their  size. 
They  stain  deep  blue  with  carbol-thionin  or  Wright's 
stain;  not  at  all  with  Ehrlich's  triple  stain.  The  cell 
containing  them  may  stain  normally  in  other  respects, 
or  it  may  exhibit  polychromatophilia.  Polychromato- 
philic  cells  generally  contain  the  smaller  granules,  which 
may  be  so  fine  that  the  cell  appears  dusted  with  them. 

Numerous  cells  showing  this  degeneration  are  com- 
monly found  in  chronic  lead-poisoning,  of  which  they 
were  at  one  time  thought  to  be  pathognomonic.  Except 
in  this  disease,  the  degeneration  indicates  a  serious  blood 
condition.  It  occurs  in  well-marked  cases  of  pernicious 
anemia  and  leukemia,  and,  much  less  commonly,  in  very 
severe  symptomatic  anemias. 

(c)  Malarial  Stippling. — This  term  has  been  applied 
to  the  finely  grariular  appearance  often  seen  in  red  cor- 
puscles, which  harbor  malarial  parasites  (see  Frontispiece^ 
PI.  VII,  and  Fig.  89).  It  was  formerly  classed  with  the 
degeneration  just  described,  but  is  undoubtedly  distinct. 
Not  all  stains  will  show  it.  With  Wright's  stain  it  can 
be  brought  out  by  staining  longer  and  washing  less  than 
for   the   ordinary   blood-stain.     The   minute   granules, 


STUDY  OF  STAINED  BLOOD 


27s 


"Schiiffner's  granules,"  stain  reddish  purple.     They  are 
sometimes  so  numerous  as  almost  to  hide  the  parasite. 


Fig.  90.— Normoblasts  from  cases  of  secondary  anemia  and  leukemia  ( X  1000)  (photo- 
graphs by  the  author). 


(3)  Variations  in  Structure. — The  most  important  is 
the  presence  of  a  nucleus  (see  Frontispiece,  PI.  IX,  and 
Fig.  90).  Nucleated  red  corpuscles,  or  erythroblasts,  are 
classed  according  to  their  size:  microblasts,  5  u  or  less  in 


Fig.  91. — Mcgaloblasts  from  a  case  of  pernicious  anemia   (X  looo)  (courtesy  of  Dr. 
W.  P.  Harlow). 

diameter;  normoblasts,  5  to  10  [i;  and  megaloblasts,  above 
10  fi.  Microblasts  and  normoblasts  contain  one,  rarely 
two,  small  round, 'sharply  defined,  deeply  staining  nuclei, 
often  located  eccentrically.    Occasionally  the  nucleus  is 


276  THE   BLOOD 

irregular  in  shape,  "clover-leaf"  forms  being  not  infre- 
quent. Sometimes  it  is  completely  broken  up  into  frag- 
ments. Mitoses  (see  Fig.  96)  are  not  uncommon  in  leu- 
kemia and  pernicious  anemia.  The  megaloblast  (Fig. 
91)  is  probably  a  distinct  cell,  not  merely  a  larger  size 
of  the  normoblast.  Its  nucleus  is  large,  stains  rather 
palely,  has  a  delicate  chromatin  network,  and  often 
shows  evidences  of  degeneration  (karyorrhexis,  etc.).  In 
ordinary  work,  however,  it  is  safer  to  base  the  distinction 
upon  size  than  upon  structure.  Any  nucleated  red  cell, 
but  especially  the  megaloblast,  may  exhibit  polychroma- 
tophilia. 

Normally,  erythroblasts  are  present  only  in  the  blood 
of  the  fetus  and  of  very  young  infants.  In  the  adult, 
their  presence  in  the  circulating  blood  denotes  an  excess- 
ive demand  upon  the  blood-forming  organs  to  regenerate 
lost  or  destroyed  red  corpuscles.  In  response  to  this 
demand,  immature  and  imperfectly  formed  cells  are 
thrown  into  the  circulation.  Their  number,  therefore, 
is  an  indication  of  the  extent  to  which  the  bone-marrow 
reacts  rather  than  of  the  severity  of  the  disease.  Nor- 
moblasts occur  in  severe  symptomatic  anemia,  leukemia, 
and  pernicious  anemia.  They  are  most  abundant  in 
myelogenous  leukemia.  While  always  present  in  per- 
nicious anemia,  they  are  often  difficult  to  find.  Megalo- 
blasts  are  found  in  pernicious  anemia,  and  with  extreme 
rarity  in  any  other  condition.  They  here  almost  inva- 
riably exceed  the  normoblasts  in  number,  which  is  one  of 
the  distinctive  features  of  the  disease.  Microblasts  have 
much  the  same  significance  as  normoblasts,  but  are 
less  common. 

Cabot's   ring  bodies  are  ring-  or  figure-of-8-shaped 


STUDY  OF  STAINED  BLOOD  277 

structures  which  have  been  observed  in  certain  of  the 
red  cells  in  pernicious  anemia,  lead-poisoning,  and  lym- 
phatic leukemia.  They  stain  red  with  Wright's  stain. 
Their  nature  is  unknown,  although  they  have  been 
thought  to  be  the  remains  of  a  nuclear  membrane. 

2.  The  Leukocytes. — An  estimation  of  the  number  or 
percentage  of  each  variety  of  leukocyte  in  the  blood  is 
called  a  differential  count.  It  probably  yields  more 
helpful  information  than  any  other  single  procedure  in 
blood  examinations. 

The  differential  count  is  best  made  upon  a  film  stained 
with  Jenner's,  Harlow's,  or  Ehrlich's  stain.  Wright's 
stain  differentiates  the  leukocytes  somewhat  less  satis- 
factorily. Go  carefully  over  the  film  with  an  oil-immer- 
sion lens,  using  a  mechanical  stage  if  available.  The 
experienced  worker  will  often  use  the  lower  powers 
(even  the  i6-mm.  objective,  as  recommended  by 
Simon)  in  routine  work.  Classify  each  leukocyte  seen, 
and  calculate  what  percentage  each  variety  is  of  the 
whole  number  classified.  For  accuracy,  500  to  1000 
leukocytes  must  be  classified;  for  approximate  results, 
300  are  sufficient.  Track  of  the  count  may  be  kept  by 
placing  a  mark  for  each  leukocyte  in  its  appropriate 
column,  ruled  upon  paper.  Some  workers  divide  a  sHde- 
box  into  compartments  with  slides,  one  for  each  variety  of 
leukocyte,  and  drop  a  coffee-bean  into  the  appropriate 
compartment  when  a  cell  is  classified.  When  a  conve- 
nient number  of  coffee-beans  is  used  (any  multiple  of 
100),  the  percentage  calculation  is  extremely  easy. 

The  actual  number  of  each  variety  in  a  cubic  milli- 
meter of  blood  is  easily  calculated  from  these  percent- 
ages and  the  total  leukocyte  count.     An  increase  in 


27B 


THE   BLOOD 


actual  number  is  an  absolute  increase;  an  increase  in  per- 
centage.only,  a  relative  increase.  It  is  evident  that  an 
absolute  increase  of  any  variety  may  be  accompanied  by 
a  relative  decrease. 

A  record  is  generally  kept  of  the  number  of  nucleated 
red  cells  seen  during  a  differential  count  of  leukocytes, 

LEUKOCYTES 

NORM/^L  /^DNORM^L 


MONONUCLE/^R  ~ 
NON  -  GR/^NUU^r? 


1-LYMPHOCYTE      20-30% 


2-ljflRGE  MONONUCLE/^Rl 


(>4-6%<  /-' 


A 


3  TR/^NSmON/^L  J 

POLYMORPHONUCLEi^f? 
GR/7NUL/7R  


MYELOCYTE 


"W   1-NEUTROPHIUC^ 


2- EOSINOPHILIC  ;« 


3-B/lSOPHILIC 


1-  NEUTPOPHILIC     60-75%  | 
2  -  EOSINOPHILIC        2-4-  %  % 

3- B>qSOPHILIC  0.3% 

P"ig.  92. — Outline  of  the  classification  of  leukocytes. 

The  usual  classification  of  leukocytes  is  based  upon 
their  size,  their  nuclei,  and  the  staining  properties  of  the 
granules  which  many  of  them  contain.  It  is  not  alto- 
gether satisfactory,  but  is  probably  the  best  which  our 
present  knowledge  permits. 

The  writer  has  found  the  table  (Fig.  92)  very  help- 


STUDY    OF   STAINED    BLOOD  279 

ful  in  impressing  this  classification  upon  the  student. 
It  makes  no  attempt  to  indicate  histogenetic  relation- 
ships. The  leukocytes  of  normal  blood  fall  into  two 
groups,  each  including  three  types.  The  cells  in  Group 
I  contain  single,  round,  oval  or  horseshoe-shaped  nuclei, 
and  have  few  or  no  granules  in  their  cytoplasm.  The 
stippling  of  the  cytoplasm  shown  in  the  diagram  repre- 
sents the  finely  granular  appearance  of  protoplasm, 
not  true  granulation.  The  cells  in  Group  II  are  polymor- 
phonuclear and  contain  granules  which  are  distinguished 
by  their  size  and  staining  reactions.  In  its  structure  the 
chief  abnormal  leukocyte,  the  myelocyte,  combines  the 
two  groups,  being  mononuclear  like  Group  I  and  gran- 
ular like  Group  11. 

(i)  Normal  Varieties. — {a)  Lymphocytes  are  small 
mononuclear  cells  without  specific  granules  (see  Frontis- 
piece and  Plate  X).  They  are  about  the  size  of  a  red 
corpuscle  or  slightly  larger  (6-10  fi),  and  consist  of  a  sin- 
gle, sharply  defined,  deeply  staining  nucleus,  surrounded 
by  a  narrow  rim  of  protoplasm.  The  nucleus  is  generally 
round,  but  is  sometimes  indented  at  one  side.  Wright's 
stain  gives  the  nucleus  a  deep  purple  color  and  the  cyto- 
plasm a  pale  robin's-egg  blue  in  typical  cells.  Larger 
lymphocytes  are  frequently  found,  especially  in  the  blood 
of  children,  and  are  difficult  to  distinguish  from  the  large 
mononuclear  leukocytes.  It  is  possible  that  the  larger 
forms  are  young  lymphocytes,  which  become  smaller  as 
they  grow  older.  In  the  cytoplasm  of  a  certain  percent- 
age of  lymphocytes  the  Romanowsky  stains  show  a  vari- 
able number  of  reddish-purple  (azurophilic)  granules. 

Lymphocytes  are  formed  in  the  lymphoid  tissues, 
including  that  of  the  bone-marrow.     They  constitute, 


28o 


THE   BLOOD 


normally,  20  to  30  per  cent,  of  all  leukocytes,  or  about 
icxDo  to  3000  per  c.mm.  of  blood.  They  are  more  abun- 
dant in  the  blood  of  children. 

The  percentage  of  lymphocytes  is  usually  moderately 
increased  in  those  conditions  which  give  leukopenia, 
especially  typhoid  fever,  chlorosis,  pernicious  anemia, 
and  many  debiUtated  conditions.  A  marked  increase, 
accompanied  by  an  increase  in  the  total  leukocyte  count, 


Fig.  93. — Lymphocytosis,  case  of  pertussis  (X  looo)  (courtesy  of  Dr.  W.  P.  Harlow). 


is  seen  in  pertussis  (Fig.  93)  and  lymphatic  leukemia.  In 
the  latter  the  lymphocytes  sometimes  exceed  98  per  cent. 
Exophthalmic  goiter  commonly  gives  a  marked  relative 
lymphocytosis,  while  simple  goiter  does  not  affect  the 
lymphocytes.  In  pulmonary  tuberculosis  a  high  per- 
centage of  lymphocytes  is  considered  a  favorable  prog- 
nostic sign. 

{b)  Large  Mononuclear  Leukocytes  (See  Frontispiece). 
— ^These  cells  are  two  or  three  times  the  diameter  of  the 


STUDY    OF   STAINED    BLOOD  28 1 

normal  red  corpuscle.  Each  contains  a  single  round  or 
oval  nucleus,  often  located  eccentrically.  The  zone  of 
protoplasm  surrounding  the  nucleus  is  relatively  wide. 
With  Wright's  stain  the  nucleus  is  less  deeply  colored 
than  that  of  the  lymphocyte,  while  the  cytoplasm  is  very 
pale  blue  or  colorless,  and  sometimes  contains  a  few  red- 
dish granules.  The  size  of  the  cell,  the  width  of  the  zone 
of  cytoplasm,  and  the  depth  of  color  of  the  nucleus  are 
the  points  to  be  considered  in  distinguishing  between 
large  mononuclears  and  lymphocytes.  When  large 
forms  of  the  lymphocyte  are  present  the  distinction  is 
often  difficult  or  impossible.  It  is  then  advisable  to 
count  the  two  cells  together  as  lymphocytes. 

Large  mononuclear  leukocytes  are  probably  devel- 
oped from  the  endothelial  cells  of  the  blood-vessels. 
They  constitute  2  to  4  per  cent,  of  the  total  number  of 
leukocytes:  100  to  400  per  c.mm.  of  blood.  An  increase 
is  unusual  except  in  malaria,  where  it  is  quite  constantly 
observed,  and  where  many  of  the  cells  contain  engulfed 
pigment. 

(c)  Transitional  Leukocytes  (See  Frontispiece). — ^These 
are  essentially  large  mononuclears  with  lobulated,  deeply 
indented,  or  horseshoe-shaped  nuclei.  A  few  fine  neu- 
trophilic granules  are  sometimes  present  in  their  cyto- 
plasm. 

They  are  probably  formed  from  the  large  mononu- 
clears, and  occur  in  the  blood  in  about  the  same  numbers. 
The  two  cells  are  counted  together,  constituting  4  to  8 
per  cent,  of  the  leukocytes. 

(d)  Polymorphonuclear  Neutrophilic  Leukocytes  (See 
Frontispiece).— There  is  usually  no  difficulty  in  recogniz- 
ing these  cells.  Their  average  size  (10  to  1 2  ^a)  is  somewhat 


282 


THE   BLOOD 


less  than  that  of  the  large  mononuclears.  The  nucleus 
stains  rather  deeply,  and  is  extremely  irregular,  often 
assuming  shapes  comparable  to  letters  of  the  alphabet, 
E,  Z,  S,  etc.  (Fig.  94).  Frequently  there  appear  to  be 
several  separate  nuclei,  hence  the  widely  used  name, 
"polynuclear  leukocyte."  Upon  careful  inspection, 
however,   delicate  nuclear  bands  connecting  the  parts 


•ft*.^^^ 


.%   ^    -^ —  * 


Fig.   94. — Marked  polymorphonuclear  neutrophilic  leukocytosis  ( X  looo)    (courtesy  of 
Dr.  W.  P.  Harlow). 

can  usually  be  seen.  The  cytoplasm  is  relatively  abun- 
dant, and  contains  great  numbers  of  very  fine  neutrophilic 
granules  (see  Fig.  97,  A).  With  Wright's  stain  the  nu- 
cleus is  bluish  purple,  and  the  granules  reddish  lilac. 

Polymorphonuclear  leukocytes  are  formed  in  the  bone- 
marrow  from  neutrophiHc  myelocytes.  They  constitute 
60  to  75  per  cent,  of  all  the  leukocytes:  3000  to  7500  per 


STUDY    OF   STAINED    BLOOD  283 

c.mm.  of  blood.  Increase  in  their  number  practically 
always  produces  an  increase  in  the  total  leukocyte  count, 
and  has  already  been  discussed  under  Polymorphonu- 
clear Leukocytosis.  The  leukocytes  of  pus,  pus-corpuscles, 
belong  almost  wholly  to  this  variety. 

A  comparison  of  the  percentage  of  polymorphonuclear 
cells  with  the  total  leukocyte  count  yields  more  informa- 
tion than  a  consideration  of  either  alone.  In  a  general 
way  the  percentage  represents  the  severity  of  the  infec- 
tion or,  more  correctly,  the  degree  of  toxic  absorption; 
while  the  total  count  indicates  the  patient's  power  of 
resistance.  With  moderate  infection  and  good  resisting 
powers  the  leukocyte  count  and  the  percentage  of  poly- 
morphonuclears are  increased  proportionately.  When 
the  polymorphonuclear  percentage  is  increased  to  a 
notably  greater  extent  than  is  the  total  number  of  leuko- 
cytes, no  matter  how  low  the  count,  either  very  poor 
resistance  or  a  very  severe  infection  may  be  inferred. 

Gibson  has  suggested  the  use  of  a  chart  to  express 
this  relationship  graphically  (Fig.  95).  Its  arrangement 
is  purely  arbitrary,  but  it  may  be  found  helpful  in  inter- 
preting counts.  An  ascending  line  from  left  to  right 
indicates  an  unfavorable  prognosis  in  proportion  as  the 
line  approaches  the  vertical.  All  fatal  cases  show  a  ris- 
ing line.  A  descending  or  horizontal  line  suggests  a  very 
favorable  prognosis. 

It  is  a  matter  of  observation  that  in  the  absence  of 
acute  infectious  disease  or  of  inflammation  directly  in 
the  blood-stream  {e.  g.,  phlebitis,  sigmoid  sinusitis,  septic 
endocarditis) ,  a  polymorphonuclear  percentage  of  85  or 
over  points  very  strongly  to  gangrene  or  pus  formation 
somewhere  in  the  body.     On  the  other  hand,  excepting 


284 


THE   BLOOD 


in  children,  where  the  percentage  is  normally  low,  pus 
is  uncommon  with  less  than  80  per  cent,  of  polymorpho- 
nuclears. 


— 

.-^o  nnci 

— 

Q-^ 

?>.5  000 

— 

— 

on 

— 

— 

^■o  onn 

— 

— 

RA 

— 

— 

1-'^  000 

= 

— 

flO 

— 

— 

10,  000 

— 

— 

73 

— 

— 

js,  000 

— 

— 

7n 

— 

— 

Total  leuko-  Percentage  of 

cyte  count.  polymorpho- 

nuclears. 
Fig.  95. — Gibson  chart  with  blood<ount  in  2  cases  of  appendicitis:  Dotted  line  rep- 
resents a  mild  case  with  prompt  recovery;  the  continuous  line,  a  very  virulent  strepto- 
coccic case  with  poor  resistance,  peritonitis,  and  early  death. 


Normally,   the  cytoplasm   of  leukocytes  stains  pale 
yellow  with  iodin.    Under  certain  pathologic  conditions 


STUDY   OF    STAINED   BLOOD  285 

the  cytoplasm  of  many  of  the  polymorphonuclears  stains 
diffusely  brown,  or  contains  granules  which  stain  reddish 
brown  with  iodin.  This  is  called  iodopkilia.  Extracellu- 
lar iodin-staining  granules,  which  are  present  normally, 
are  more  numerous  in  iodophilia. 

This  iodin  reaction  occurs  in  all  purulent  conditions 
except  abscesses  which  are  thoroughly  walled  off  and 
purely  tuberculous  abscesses.  It  is  of  some  value  in 
diagnosis  between  serous  effusions  and  purulent  exu- 
dates, between  catarrhal  and  suppurative  processes  in 
the  appendix  and  Fallopian  tube,  etc.  Its  importance, 
however,  as  a  diagnostic  sign  of  suppuration  has  been 
much  exaggerated,  since  it  may  occur  in  any  general 
toxemia,  such  as  pneumonia,  influenza,  maUgnant  dis- 
ease, and  puerperal  sepsis. 

To  demonstrate  iodophilia,  place  the  air-dried  films  in 
a  stoppered  bottle  containing  a  few  crystals  of  iodin  until 
the  films  become  yellow.  Mount  in  syrup  of  levulose 
and  examine  with  an  immersion  objective. 

Arneth  classifies  polymorphonuclear  leukocytes  into 
five  groups,  according  to  the  number  of  lobes  which  the 
nucleus  shows.  The  percentage  of  cells  in  each  group 
is  fairly  constant  in  health,  but  shows  considerable  varia- 
tion in  disease. 

{e)  Eosinophilic  Leukocytes,  or  "  Eosinophiles "  (See 
Frontispiece). — The  structure  of  these  cells  is  similar  to 
that  of  the  polymorphonuclear  neutrophiles,  with  the 
striking  difference  that,  instead  of  fine  neutrophihc  gran- 
ules, their  cytoplasm  contains  coarse  round  or  oval  gran- 
,ules  having  a  strong  affinity  for  acid  stains.  They  are 
easily  recognized  by  the  size  and  color  of  the  granules, 
which  stain  bright  red  with  stains  containing  eosin  (see 


286  THE   BLOOD 

Fig.  97,  B).  Their  cytoplasm  has  generally  a  faint  sky- 
blue  tinge,  and  the  nucleus  stains  somewhat  less  deeply 
than  that  of  the  polymorphonuclear  neutrophile.     . 

Eosinophiles  are  formed  in  the  bone-marrow  from 
eosinophilic  myelocytes.  Their  normal  number  varies 
from  50  to  400  per  c.mm.  of  blood,  or  i  to  4  per  cent,  of 
the  leukocytes.  An  increase  is  called  eosinopMlia,  and  is 
better  determined  by  the  actual  number  than  by  the 
percentage. 

Slight  eosinophilia  is  physiologic  during  menstruation. 
Marked  eosinophilia  is  always  pathologic.  It  occurs  in 
a  variety  of  conditions,  the  most  important  of  which  are: 
infection  by  animal  parasites;  bronchial  asthma;  mye- 
logenous leukemia;  scarlet  fever,  and  many  skin  diseases. 

(a)  Eosinophilia  may  be  a  symptom  of  infection  by 
any  of  the  worms.  It  is  fairly  constant  in  trichiniasis, 
uncinariasis,  filariasis,  and  echinococcus  disease.  In  this 
country  an  unexplained  marked  eosinophiha  warrants 
examination  of  a  portion  of  muscle  for  Trichinella  spiralis 
(see  p.  415).  The  cells  usually  range  between  10  and  30 
per  cent,  of  all  the  leukocytes,  but  may  go  much  higher. 

(b)  True  bronchial  asthma  commonly  gives  a  marked 
eosinophilia  during  and  following  the  paroxysms.  This 
is  helpful  in  excluding  asthma  of  other  origin.  Eosino- 
philes also  appear  in  the  sputum  in  large  numbers. 

ic)  In  myelogenous  leukemia  there  is  almost  invariably 
an  absolute  increase  of  eosinophiles,  although,  owing  to 
the  great  increase  of  other  leukocytes,  the  percentage  is 
usually  diminished.  Dwarf  and  giant  forms  are  often 
numerous. 

id)  Scarlet  fever  is  frequently  accompanied  by  eosino- 
philia, which  may  help  to  distinguish  it  from  measles. 


STUDY   OF   STAINED   BLOOD 


287 


(e)  Eosinophilia  has  been  observed  in  a  large  number 
of  skin  diseases,  notably  pemphigus,  prurigo,  psoriasis, 
and  urticaria.  It  probably  depends  less  upon  the  variety 
of  the  disease  than  upon  its  extent. 

(/)  Basophilic  Leukocytes  or  "  Mast-cells  "  (See  Fron- 
tispiece).— In  general,  these  resemble  polymorphonuclear 
neutrophiles  except  that  the  nucleus  is  less  irregular  (usu- 
ally merely  indented  or  slightly  lobulated)  and  that  the 
granules  are  larger  and  have  a  strong  affinity  for  basic 


Fig.  96. — Basophilic  leukocytes.    At  the  right  is  also  a  normoblast  undergoing  mitosis 
(X  1000)  (photographs  by  the  author). 


stains.  They  are  easily  recognized  (Figs.  96  and  97,  C). 
With  Wright's  stain  the  granules  are  deep  purple,  while 
the  nucleus  is  pale  blue  and  is  often  nearly  or  quite  hid- 
den by  the  granules,  so  that  its  form  is  difficult  to  make 
out.  These  granules  are  not  colored  by  EhrHch's  stain. 
Mast-cells  probably  originate  in  the  bone-marrow  from 
basophilic  myelocytes.  They  are  least  numerous  of  the 
leukocytes  in  normal  blood,  rarely  exceeding  0.5  per 
cent.,  or  25  to  50  per  c.mm.    A  notable  increase  is  lim- 


288 


THE  BLOOD 


ited  almost  exclusively  to  myelogenous  leukemia,  where 
they  are  sometimes  very  numerous. 


ABC 

Fig.  97. — Ruptured  leukocytes,  showing  relative  size  of  Rranules:  A,  neutrophilic;  B, 
eosinophilic;  C,  basophilic  (Xiooo)  (photographs  by  the  author). 

(2)  Abnormal  Varieties.^ — (a)  Myelocytes  (see  Fron- 
tispiece and  Fig.  98)  are  large  mononuclear  cells  whose 


A  B 

Fig.  98. — Myelocytes  from  blood  of  myelogenous  leukemia:   A,  Neutrophilic;  B,  eosino- 
philic (Xiooo)  (photographs  by  the  author). 

cytoplasm  is  filled  with  granules.    Typically,  the  nucleus 
occupies  about  one-half  of  the  cell,  and  is  round  or  oval. 


STUDY  OF  STAINED  BLOOD  289 

It  is  sometimes  indented,  with  its  convex  side  in  contact 
with  the  periphery  of  the  cell.  It  stains  rather  feebly. 
The  average  diameter  of  this  cell  (about  15.75  fj.)  is 
greater  than  that  of  any  other  leukocyte,  but  there  is 
much  variation  in  size  among  individual  cells.  Myelo- 
cytes are  named  according  to  the  character  of  their 
granules — neutrophilic,  eosinophilic,  and  basophihc  mye- 
locytes. These  granules  are  identical  with  the  corres- 
ponding granules  in  the  leukocytes  just  described.  They 
are,  however,  often  less  distinct  and  less  sharply  differ- 
entiated by  the  various  stains  than  those  of  the  corres- 
ponding polymorphonuclear  cells.  In  some  the  granules 
are  few  in  number,  the  cells  departing  but  little  from  the 
structure  of  the  parent  myeloblast.  Although  the 
occurrence  of  two  kinds  of  granules  in  the  same  cell  is 
rare,  a  few  basophilic  granules  are  sometimes  seen  in 
young  eosinophilic  myelocytes.  The  basophilic  myelocyte 
is  usually  small ;  and  its  nucleus  is  commonly  so  pale  and 
so  obscured  by  the  granules  that  the  cell  is  not  easily 
distinguished  from  the  mast-cell. 

Myelocytes  are  the  bone-marrow  cells  from  which  the 
corresponding  granular  leukocytes  are  developed.  They 
in  turn  are  derived  from  non-granular  cells  of  the  bone- 
marrow,  the  myeloblasts.  Their  presence  in  the  blood 
in  considerable  numbers  is  diagnostic  of  myelogenous 
leukemia.  The  neutrophilic  form  is  the  least  significant. 
A  few  of  these  may  be  present  in  very  marked  leukocy- 
tosis or  any  severe  blood  condition,  as  pernicious  anemia. 
Eosinophilic  myelocytes  are  found  only  in  myelogenous 
leukemia,  where  they  are  often  very  numerous.  The 
basophilic  variety  is  less  common,  and  is  confined  to 
long-standing,  severe  myelogenous  leukemia. 

19 


290  THE   BLOOD 

(b)  Myeloblasts. — ^These  are  the  parent  cells  of  the 
myelocytes,  from  which  they  differ  chiefly  in  the  ab- 
sence of  cytoplasmic  granules.  Their  round  or  oval 
nuclei  contain  several  nucleoli.  The  cytoplasm  is  some- 
what basophilic. 

Myeloblasts  appear  in  the  blood  in  considerable 
numbers  in  acute  myelogenous  leukemia  and  the  ter- 
minal stages  of  chronic  myelogenous  leukemia,  when 
the  bone-marrow  reverts  to  the  embryonic  t^pe.  They 
may  be  indistinguishable  morphologically  from  the  large 
lymphocytes  of  acute  lymphatic  leukemia,  but  can 
usually  be  distinguished  by  the  oxydase  reaction. 

Indophenol  Oxydase  Test. — Fix  cover-glass  films  in  alco- 
hol for  five  minutes.  Float  the  covers  for  ten  to  twenty 
minutes  face  downward  upon  a  freshly  prepared  solution 
containing  equal  parts  of  i  per  cent,  aqueous  solutions  of 
dimethyl-para-phenylendiamin  and  of  alpha-naphthol.  Rinse 
in  water  and  mount  in  water,  glycerin,  or  a  strong  solution 
of  water-glass. 

The  cytoplasm  of  cells  containing  oxydase — pol>Tnorpho- 
nuclears,  large  mononuclears,  myeloc\'tes,  and  myeloblasts 
— will  be  colored  pale  blue  to  dark  blue.  Lymphocytes, 
red  corpuscles,  and  plaques  should  show  no  blue  whatever. 

(c)  Atjrpic  Forms. — Leukocytes  which  do  not  fit  in 
with  the  above  classification  are  not  infrequently  met, 
especially  in  high-grade  leukocytosis,  pernicious  anemia, 
and  leukemia.  The  nature  of  many  of  them  is  not 
clear,  and  their  number  is  usually  so  small  that  they 
may  be  disregarded  in  making  a  differential  count. 
Among  them  are: 

(a)  Border-line  forms  between  polymorphonuclear 
neutrophiles  and  neutrophilic  myelocytes. 


STUDY  OF  STAINED  BLOOD  29 1 

(b)  Small  neutrophilic  cells  with  a  single  round, 
deeply  staining  nucleus;  they  probably  result  from  di- 
vision of  polymorphonuclear  neutrophiles. 

(c)  "Irritation  forms" — large  non-granular  mononu- 
clear cells,  whose  cytoplasm  stains  fairly  deep  purple 
with  Wright's  stain  and  intense  brown  with  EhrHch's, 
and  usually  contains  vacuoles.  They  appear  in  the 
blood  under  the  same  conditions  as  myelocytes. 

(d)  Degenerated  forms:  vacuolated  leukocytes,  or 
merely  palely  or  deeply  staining  homogeneous  or  retic- 


Fig.  99. — A  cluster  of  blood-plaques  and  two  plaques  lying  up)on  a  red  cell  and  simu- 
lating malarial  parasites  (Xiooo)  ^photograph  by  the  author). 


ulated  masses  of  chromatin  (the  so-caUed  "basket-cells," 
see  Frontispiece). 

3.  Blood=plaques. — These  are  not  colored  by  EhrHch's 
stain  nor  by  eosin  and  methylene-blue.  With  Wright's 
stain  they  appear  as  spheric  or  ovoid,  reddish  to  violet, 
granular  bodies,  2  to  4  u  in  diameter.  Occasionally  a 
platelet  as  large  as  a  red  corpuscle  is  seen.  When  well 
-stained  a  deHcate  hyaline  peripheral  zone  can  be  dis- 
tinguished. In  ordinary  blood-smears  they  are  usually 
clumped  in  masses.    A  single  platelet  lying  upon  a  red 


292  THE   BLOOD 

corpuscle  may  easily  be  mistaken  for  a  malarial  parasite 
(see  Frontispiece  and  Fig.  99). 

Blood-platelets  are  being  much  studied  at  present,  but, 
aside  from  the  facts  mentioned  under  their  enumeration 
(see  p.  258),  little  of  clinical  value  has  been  learned.  They 
have  been  variously  regarded  as  very  young  red  corpus- 
cles (the  "hematoblasts"  of  Hayem),  as  disintegration 
products  of  leukocytes,  as  remnants  of  extruded  nuclei 
of  erythrocytes,  and  as  independent  nucleated  bodies. 
The  most  probable  explanation  of  their  origin  seems  to 
be  that  of  J.  H.  Wright,  who  regards  them  as  detached 
portions  of  the  cytoplasm  of  certain  giant-cells  of  the 
bone-marrow  and  spleen. 

K.    BLOOD  PARASITES 
A.   Bacteria 

Bacteriologic  study  of  the  blood  is  useful  in  many 
conditions,  but  in  general,  the  elaborate  technic  involved 
takes  it  out  of  the  reach  of  the  clinician.  As  applied  to 
the  diagnosis  of  typhoid  fever,  however,  the  technic  of 
blood-cultures  has  been  so  simplified  that  it  can  be  car- 
ried through  by  any  one  who  is  competent  to  do  the 
simplest  cultural  work. 

Typhoid  bacilli  can  be  detected  in  the  blood  in  prac- 
tically every  case  of  typhoid  fever  in  the  first  week  of  the 
disease;  in  about  80  to  85  per  cent,  of  cases  in  the  second 
week;  and  in  decreasing  percentages  in  the  later  weeks. 
The  blood-culture,  therefore,  offers  the  most  certain 
means  of  early  diagnosis.  It  is  in  a  sense  complementary 
to  the  Widal  reaction,  the  former  decreasing  and  the 
latter  increasing  in  rehability  as  the  disease  progresses. 


BLOOD   PARASITES  293 

The  blood-culture  gives  best  results  before  the  Widal 
appears,  as  one  would  expect  from  the  fact  that  the 
Widal  test  depends  upon  the  presence  of  antibodies 
which  destroy  or,  at  least,  injure  the  bacilH.  The  two 
methods  together  will  estabhsh  the  diagnosis  in  prac- 
tically every  case  at  any  stage.  BacilU  disappear  from 
the  blood  in  convalescence  and  reappear  in  a  relapse. 

Technic  of  Blood-cultures  in  Typhoid  Fever. — ^The  blood 
may  be  obtained  in  one  of  two  ways : 

(a)  With  a  spring-lancet  make  a  deep  puncture  in  the 
edge  (not  the  side)  of  the  lobe  of  the  ear,  as  for  a  blood-count. 
Allow  the  blood  to  drop  directly  into  a  short  culture-tube 
containing  the  bile  medium.  By  gentle  milking,  20  to  40 
drops  can  usually  be  obtained.  This  simple  method  of  ob- 
taining blood  is  especially  applicable  dvuing  the  first  week  of 
the  disease  when  .bacilli  are  abundant.  Contamination  with 
skin  cocci  is  possible,  but  does  not  usually  interfere  when  the 
bile  mediiun  is  used. 

(b)  In  the  later  weeks  of  the  disease  a  larger  quantity  of 
blood  is  needed.  Prepare  the  skin  on  the  front  of  the  elbow 
as  for  a  minor  operation,  or  simply  rub  well  with  alcohol  or 
paint  with  tincture  of  iodin.  Tie  a  bandage  tightly  around 
the  upper  arm,  have  the  patient  open  and  close  the  fist  a  few 
times,  and  when  the  veins  are  sufficiently  distended  insert 
a  hypodermic  needle  attached  to  a  syringe  into  any  vein  that 
is  prominent.  The  needle  should  go  through  the  skin  about 
J  inch  from  the  vein  with  the  bevel  at  its  tip  uppermost,  and 
should  enter  the  vein  from  the  side  in  a  direction  opposite 
to  the  blood-current  (Fig.  100).  Unless  too  smaU  a  needle 
is  used,  blood  will  begin  to  rise  in  the  syringe  as  soon  as  the 
needle  has  entered  the  vein.  Suction  is  rarely  necessary. 
When  sufficient  blood  is  obtained,  the  bandage  is  first  re- 
moved, the  needle  is  withdrawn,  and  the  blood  is  allowed  to 


294 


THE    BLOOD 


run  into  a  tul^e  of  culture-medium.  It  is  usually  easy  to 
secure  5  to  10  c.c.  of  blood.  The  procedure  causes  the  patient 
surprisingly  little  inconvenience,  seldom  more  than  does  an 
ordinary-  hypodermic  injection.  There  is  rarely  any  difficulty 
in  entering  the  vein  except  in  children,  and  in  adults  when  the 
arm  is  fat  and  the  veins  are  small.  If  desired,  one  of  the 
veins  about  the  ankle  can  be  used.  Instead  of  a  syringe  one 
can  use  a  large  glass  tube  which  has  been  drawn  out  at  the 
ends  and  one  end  ground  to  fit  a  ''slip-on"  needle.  Either 
a  large  hypodermic  needle  or  a  small  antitoxin  needle  may 


Fig.  100. — Method  of  obtaining  blood  for  a  blood-culture. 


be  used.  These  little  instruments  (Fig.  ico)  can  be  made  by 
any  glass-blower  at  a  cost  of  about  fifty  cents,  and  several 
of  them  can  be  kept  on  hand  in  test-tubes  sterilized  ready  for 
use. 

As  special  culture-medium,  ox-bile  is  generally  used.  It 
favors  the  growth  of  the  t\phoid  bacillus  and  retards  the 
growth  of  other  organisms.   A  good  formula  is  given  on  p.  463. 

As  soon  as  convenient  after  the  blood  is  added,  place  the 
tubes  in  the  incubator.  After  about  twelve  hours  examine 
for  motile  bacilli.  If  none  are  found,  transfer  a  few  drops 
to  tubes  of  bouillon  or  solidified  blood-serum  and  incubate  for 


BLOOD   PARASITES  295 

twelve  hours  longer.  If  motile,  Gram-negative  bacilli  are 
found;  they  are  almost  certainly  typhoid  baciUi.  Further 
study  is,  however,  desirable,  especially  from  a  scientific 
point  of  view.  The  only  bacilli  which  might  cause  confusion 
are  the  paratyphoid  and  colon  bacilli,  which  can  be  distin- 
guished by  gas  production  in  glucose  media,  indol  produc- 
tion, and  their  effect  upon  litmus  milk  (see  p.  462).  The 
agglutination  test  for  the  identity  of  the  bacillus  is  not  avail- 
able clinically,  since  freshly  isolated  bacilli  do  not  agglutinate 
well. 

B.   Animal  Parasites 

Of  the  animal  parasites  which  have  been  found  in  the 
blood,  five  are  interesting  clinically:  the  spirochete  of 
relapsing  fever;  trypanosomes;  malarial  parasites;  filarial 
larvae;  and  the  larva?  of  Trichinella  spiralis. 

1.  Spirochaeta  recurrentis  is  described  on  p.  373, 

2.  Trypanosoma  gambiense. — Various  trypanosomes 
are  common  in  the  blood  of  fishes,  amphibians,  birds,  and 
mammals  (see  Fig.  115).  They  live  in  the  blood-plasma 
and  do  not  attack  the  corpuscles.  In  some  animals  they 
are  apparently  harmless ;  in  others  they  are  an  important 
cause  of  disease.    They  are  discussed  more  fully  on  p.  376. 

The  trypanosome  of  human  blood,  Trypanosoma  gam- 
biense (Plate  VII),  is  an  actively  motile,  spindle-shaped 
organism,  two  or  three  times  the  diameter  of  a  red  cor- 
puscle in  length,  with  an  undulating  membrane  which 
terminates  at  the  anterior  end  in  a  long  flagellum.  It 
can  be  seen  with  medium-power  objectives  in  fresh  blood, 
but  is  best  studied  with  an  oil-immersion  lens  in  prepara- 
tions stained  as  for  the  malarial  parasite.  It  may  be 
necessary  to  search  many  slides.  Human  trypano- 
somiasis is  common  in  Africa.     As  a  rule,  it  is  a  very 


296  THE   BLOOD 

chronic  disease.  "Sleeping  sickness"  is  a  late  stage  when 
the  organisms  have  invaded  the  cerebrospinal  fluid. 
Infection  is  carried  by  the  tsetse  fly,  Glossina  palpalis. 

3.  The  Malarial  Parasites. — These  protozoa  belong  to 
the  Sporozoa  (see  p.  381),  order  Hemosporidia,  the  mem- 
bers of  which  are  parasites  in  the  blood  of  a  great  variety 
of  vertebrates.  Three  species,  constituting  the  genus 
Plasmodium,  are  associated  with  malarial  fever  in  man: 
Plasmodium  vivax,  P.  malaria,  and  P.  falciparum,  the 
parasites  respectively  of  the  tertian,  quartan,  and 
estivo-autumnal  t>^es  of  malaria.^  The  life  histories  of 
the  three  are  so  similar  that  they  may  well  be  described 
together. 

(i)  Life  Histories. — There  are  two  cycles  of  develop- 
ment: one,  the  asexual,  in  the  blood  of  man;  and  the 
other,  the  sexual,  in  the  intestinal  tract  of  a  particular 
genus  of  mosquito.  Anopheles. 

{a)  Asexual  Cycle. — The  young  organism  enters  the 
blood  through  the  bite  of  the  mosquito.  It  makes  its  way 
into  a  red  corpuscle,  where  it  appears  as  a  small,  pale, 
"hyaline"  body.  This  body  exhibits  ameboid  move- 
ment and  increases  in  size.  Soon  dark-brown  granules, 
derived  from  the  hemoglobin  of  the  corpuscle,  make  their 
appearance  within  it.  When  it  has  reached  its  full  size 
— filling  and  distending  the  corpuscle  in  the  case  of  the 
tertian  parasite,  smaller  in  the  others — the  pigment 
granules  gather  at  the  center  or  at  one  side;  the  organ- 
ism divides  into  a  number  of  small  hyaline  bodies,  the 
spores  or  merozoites;  and  the  red  corpuscle  bursts, 
setting  spores  and  pigment  free  in  the  blood-plasma. 

'  A  new  species,  occurring  in  India,  has  very  recently  been  described 
by  Stephens.    The  name  Plasmodium  tenue  has  been  given. 


PLATE   Vll 


ivV*ii 


Trypanosoma  gambiense  (slide  presented  by  Professor  F.  G.  Novy). 


Tertian  malarial  parasites,  one  red  Estivo-autumnal  malarial  para- 

cell  showing  malarial  stippling.  sites,     small     ring     forms      and 

crescents. 


Spirochaeta  novyi. 
Animal  parasites  of  the  blood;   X  looo  (photographs  by  the  author). 


BLOOD   PARASITES  297 

This  is  called  segmentation.  It  coincides  with,  and  by 
Hberation  of  toxins  causes,  the  paroxysm  of  the  disease. 
A  considerable  number  of  the  spores  are  destroyed  by 
leukocytes  or  other  agencies;  the  remainder  enter  other 
corpuscles  and  repeat  the  cycle.  Many  of  the  pigment 
granules  are  taken  up  by  leukocytes.  Migration  of  young 
parasites  from  corpuscle  to  corpuscle  between  parox- 
ysms has  recently  been  noted.  In  estivo-autumnal 
fever  segmentation  occurs  in  the  internal  organs  and  the 
segmenting  and  larger  pigmented  forms  are  not  seen  in 
the  peripheral  blood. 

The  asexual  cycle  of  the  tertian  organism  occupies 
forty-eight  hours;  of  the  quartan,  seventy- two  hours;  of 
the  estivo-autumnal,  an  indefinite  time — usually  twenty- 
four  to  forty-eight  hours. 

The  parasites  are  thus  present  in  the  blood  in  great 
groups,  all  the  individuals  of  which  reach  maturity  and 
segment  at  approximately  the  same  time.  This  explains 
the  regular  recurrence  of  the  paroxysms  at  intervals  cor- 
responding to  the  time  occupied  by  the  asexual  cycle  of 
the  parasite.  Not  infrequently  there  is  multiple  infection, 
one  group  reaching  maturity  while  the  others  are  still 
young;  but  the  presence  of  two  groups  which  segment 
upon  the  same  day  is  extremely  rare.  Fevers  of  longer 
intervals — six,  eight,  ten  days — are  probably  due  to  the 
ability  of  the  body,  sometimes  of  itself,  sometimes  by  aid 
of  quinin,  to  resist  the  parasites,  so  that  numbers  suffi- 
cient to  cause  a  paroxysm  do  not  accumulate  in  the 
blood  until  after  several  repetitions  of  the  asexual  cycle. 
In  estivo-autumnal  fever  the  regular  grouping,  while  usu- 
ally present  at  first,  is  soon  lost,  thus  causing  "irregular 
malaria." 


298  THE    BLOOD 

Bass  has  recently  succeeded  in  cultivating  the  malarial 
parasite  outside  of  the  body. 

(b)  Sexual  Cycle. — Besides  the  ameboid  individuals 
which  pass  through  the  asexual  cycle,  there  are  present 
with  them  in  the  blood  many  individuals  with  sexual 
properties.  These  are  called  gametes.  The  female  is 
a  little  the  larger.  The  gametes  do  not  undergo  seg- 
mentation, but  grow  to  adult  size  and  remain  inactive 
in  the  blood  until  taken  up  by  a  mosquito.  Many  of 
them  are  apparently  extracellular,  but  stained  prepara- 
tions usually  show  them  to  be  surrounded  by  the  re- 
mains of  a  corpuscle.  In  tertian  and  quartan  malaria 
they  cannot  easily  be  distinguished  from  the  asexual 
individuals  until  a  variable  time  after  the  blood  leaves 
the  body,  when  the  male  gamete  sends  out  one  or  more 
flagella.  In  estivo-autumnal  malaria  the  gametes  take 
distinctive  ovoid  and  crescentic  forms,  and  are  not 
difficult  to  recognize.  They  are  very  resistant  to  quinin 
and  often  persist  in  the  blood  long  after  the  ameboid 
forms  have  been  destroyed,  but  are  probably  incapable 
of  continuing  the  disease  until  they  have  passed  through 
the  cycle  in  the  mosquito. 

When  a  malarious  person  is  bitten  by  a  mosquito,  the 
gametes  are  taken  with  the  blood  into  its  stomach. 
Here  the  male  sends  out  one  or  more  flagella.  These 
break  off  and  seek  out  the  females,  which  they  penetrate 
much  as  the  sperm  penetrates  the  ovum.  The  female 
soon  thereafter  becomes  encysted  in  the  wall  of  the  intes- 
tine. After  a  time  it  ruptures,  liberating  many  minute 
rods,  or  sporozoites,  which  have  formed  within  it.  These 
migrate  to  the  salivary  glands,  and  are  carried  into  the 
blood  of  the  person  whom  the  mosquito  bites.    Here  they 


BLOOD   PARASITES 


299 


enter  red  corpuscles  as  young  malarial  parasites,  and  the 
majority  pass  through  the  asexual  cycle  just  described. 
The  sexual  cycle  can  take  place  only  within  the  body 
of  the  female  of  one  genus  of  mosquito,  Anopheles. 
The  male  does  not  bite.  Absence  of  this  mosquito  from 
certain  districts  explains  the  absence  of  malaria.  It  is 
distinguished  from  our  common  house-mosquito,  Culex, 
by  the  relative  lengths  of  proboscis  and  palpi  (Fig.  loi), 
which  can  be  seen  with  a  hand-lens,  by  its  attitude  when 


Fig.  loi. — Mosquitoes:  i,  Culex;  2.  Anoplieles  (Bergey). 


resting,  and  by  its  dappled  wing  (Fig.  102).  Anopheles 
is  strictly  nocturnal  in  its  habits ;  it  usually  flies  low,  and 
rarely  travels  more  than  a  few  hundred  yards  from  its 
breeding-place,  although  it  may  be  carried  by  winds. 
These  facts  explain  certain  peculiarities  in  malarial  in- 
fection; thus,  infection  occurs  practically  only  at  night; 
it  is  most  common  near  stagnant  water,  especially  upon 
the  side  toward  which  the  prevailing  winds  blow;  and  the 
danger  is  greater  when  persons  sleep  upon  or  near  the 
ground  than  in  upper  stories  of  buildings.     The  insects 


300 


THE   BLOOD 


frequently  hibernate  in  warmed  houses,  and  may  bite 
during  the  winter,  A  mosquito  becomes  dangerous  in 
eight  to  fourteen  days  after  it  bites  a  malarious  person, 
and  remains  so  throughout  its  life. 


Fig.  I02. — Showing,  on  the  left,  Anopheles  in  resting  position,  its  dappled  wing,  and 
the  position  of  its  larva?  in  water;  on  the  right,  Culex  in  resting  position,  its  plain  wing, 
and  the  position  of  its  larvae  in  water.  The  arrows  indicate  the  directions  taken  by  the 
larvae  when  the  water  is  disturbed  (Abbott). 


(2)  Detection. — Search  for  the  malarial  parasite  may 
be  made  in  either  fresh  blood  or  stained  films.  If  pos- 
sible, the  blood  should  be  obtained  a  few  hours  before 
the  chill — never  during  it  nor  within  a  few  hours  after- 
ward, since  at  that  time  (in  single  infections)  only  the 
very  young,  unpigmented  forms  are  present,  and  these 


BLOOD   PARASITES  3OI 

are  the  most  difficult  to  find  and  recognize.  Sometimes 
many  parasites  are  found  in  a  microscopic  field ;  some- 
times, especially  in  estivo-autumnal  infection,  owing  to 
accumulation  in  internal  organs,  careful  search  is  required 
to  find  any,  despite  very  severe  symptoms.  Quinin  causes 
them  rapidly  to  disappear  from  the  peripheral  blood, 
and  few  or  none  may  be  found  after  its  administration. 
In  the  absence  of  organisms,  the  presence  of  pigment 
granules  within  leukocytes — especially  the  large  mono- 
nuclears— may  be  taken  as  presumptive  evidence  of 
malaria.  Pigmented  leukocytes  (see  Frontispiece)  are 
most  munerous  after  a  paroxysm. 

(a)  In  Fresh  Unstained  Blood  (Plate  VIII) . — Obtain  a 
small  drop  of  blood  from  the  finger  or  lobe  of  the  ear. 
Touch  the  center  of  a  cover-glass  to  the  top  of  the  drop 
and  quickly  place  it,  blood  side  down,  upon  a  slide.  If 
the  shde  and  cover  be  perfectly  clean  and  the  drop  not 
too  large,  the  blood  will  spread  out  so  as  to  present  only 
one  layer  of  corpuscles.  Search  with  an  oil-immersion 
objective,  using  very  subdued  light.  The  preparation 
may  be  kept  for  many  hours  if  ringed  with  vaseHn  or 
melted  paraffin. 

The  young  organisms  appear  as  small,  round,  ring-Uke 
or  irregular,  colorless  bodies  within  red  corpuscles.  The 
hght  spots  caused  by  crenation  and  other  changes  in  the 
corpuscles  are  frequently  mistaken  for  them,  but  are 
generally  more  refractive  or  have  more  sharply  defined 
edges.  The  older  forms  are  larger  colorless  bodies  con- 
taining granules  of  brown  pigment.  In  the  case  of  the 
tertian  parasite,  these  granules  have  active  vibratory 
motion,  which  renders  them  conspicuous;  and  as  the 
parasite  itself  is  very  pale,  one  may  see  only  a  large  pale 


302  THE   BLOOD 

corpuscle  in  which  tine  pigment  granules  are  dancing. 
Segmenting  organisms,  when  typic,  appear  as  rosets, 
often  compared  to  daisies,  the  petals  of  which  represent 
the  segments,  while  the  central  brown  portion  represents 
the  pigment.  Tertian  segmenting  forms  are  less  fre- 
quently tv-pic  than  quartan.  Flagellated  forms  are  not 
seen  until  ten  to  twenty  minutes  after  the  blood  has  left 
the  vessels.  As  Cabot  suggests,  one  should,  while  search- 
ing, keep  a  sharp  lookout  for  unusually  large  or  pale  cor- 
puscles, and  for  anything  which  is  brown  or  black  or  in 
motion. 

(b)  In  Stained  Films  (See  Frontispiece  and  Plate  VII). 
— Recognition  of  the  parasite,  especially  the  young  forms, 
is  much  easier  in  lilms  stained  by  Wright's  or  some  simi- 
lar stain  than  in  fresh  blood.  The  films  must  be  well 
stained.  It  is  useless  to  search  preparations  in  which  the 
nuclei  of  leukocytes  are  very  pale.  When  parasites  are 
very  scarce,  they  may  sometimes  be  found,  although  their 
structure  is  not  well  shown  by  the  method  of  Ruge. 
This  consists  in  spreading  a  very  thick  layer  of  blood, 
drying,  placing  for  a  few  minutes  in  a  fluid  containing 
5  per  cent,  formalin  and  i  per  cent,  acetic  acid,  which 
removes  the  hemoglobin  and  fixes  the  smear,  rinsing,  dry- 
ing, and  fmally  staining.  Carbol-thionin  is  very  useful 
for  this  purpose.  If  Wright's  stain  be  used  in  this 
method,  it  is  recommended  that  the  preparation  be 
subsequently  stained  for  a  half-minute  with  borax- 
methylene-blue  (borax,  5;  methylene-blue,  2;  water,  100). 

In  films  which  are  properly  stained  with  Wright's  fluid 
the  young  organisms  are  small,  round,  ring-like  or  irreg- 
ular, sky-blue  bodies,  each  with  a  ver>'  small,  sharply  de- 
fined, reddish-purple  chromatin  mass.    Many  structures 


inv  __,_ 


i!igiq  bsqolavafa  vilui  ,|.i 
iih  .OS  ,pi  ,8i  ;fmul  3JbJIj;j 
gnuaoragaa  ,|.s  ,£s  jeraioi  faaJnanjgiq  , . 


Explanation  of  Plate  VIII 

Various  forms  of  malarial  parasites  (unstained)  (Thayer  and  Hewetson). 
I  to  lo,  inclusive,  Tertian  organisms;  ii  to  17,  inclusive,  quartan 
organisms;  18  to  27,  inclusive,  estivo-auturanal  organisms;  i,  young 
hyaline  form;  2,  hyaline  form  with  beginning  pigmentation;  3,  pig- 
mented form;  4,  full-grown  pigmented  form;  5,  6,  7,  8,  segmenting 
forms;  9,  extracellular  pigmented  form;  10,  flagellate  form;  11,  young 
hyaline  form;  12,  13,  pigmented  forms;  14,  fully  developed  pigmented 
form;  15,  16,  segmenting  forms;  17,  flagellate  form;  18,  19,  20,  ring-like 
and  cross-like  hyaline  forms;  21,  22,  pigmented  forms;  23,  24,  segmenting 
forms;  25,  26,  27,  crescents. 


PLATE   VIII 


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M 


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JS 


J6 


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IS 


20 


21 


22 


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24^ 


25 


26 


27 


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■<t, 


BLOOD   PARASITES  303 

— deposits  of  stain,  dirt,  blood-plaques  lying  upon  red 
cells  (see  Fig.  99),  etc. — may  simulate  them,  but  should 
not  deceive  one  who  looks  carefully  for  both  the  blue 
cytoplasm  and  the  reddish-purple  chromatin.  A  plaque 
upon  a  red  corpuscle  is  surrounded  by  a  colorless  zone 
rather  than  by  a  distinct  blue  body.  Young  estivo- 
autumnal  parasites  commonly  take  a  "ring"  form 
(the  chromatin  mass  representing  the  jewel),  which  is 
infrequently  assumed  by  the  other  varieties.  The  older 
tertian  and  quartan  organisms  show  larger  sky-blue 
bodies  with  more  reticular  chromatin,  and  contain 
brown  granules  of  pigment,  which,  however,  is  less 
evident  than  in  the  hving  parasite.  The  chromatin  is 
often  scattered  through  the  cytoplasm  or  apparently 
outside  of  it,  and  is  sometimes  difficult  to  see  clearly. 
Typical  "segmenters"  present  a  ring  of  rounded  seg- 
ments or  spores,  each  with  a  small,  dot-Uke  chromatin 
mass.  With  the  tertian  parasite,  the  segments  more  fre- 
quently form  an  irregular  cluster.  The  pigment  is  col- 
lected near  the  center  or  scattered  among  the  segments. 
In  estivo-autumnal  fever  usually  only  the  small  "ring 
bodies"  and  the  crescentic  and  ovoid  gametes  are  seen 
in  the  blood.  The  gametes  are  easily  recognized.  Their 
length  is  somewhat  greater  than  the  diameter  of  a  red 
corpuscle.  Their  chromatin  is  usually  centrally  placed, 
and  they  contain  more  or  less  coarse  pigment.  The  re- 
mains of  the  red  cell  often  form  a  narrow  rim  around  them 
or  fill  the  concavity  of  the  crescent. 

While  the  parasites  are  more  easily  found  in  stained 
preparations,  the  varieties  are  more  easily  differentiated 
in  fresh  blood.  The  chief  distinguishing  points  are 
included  in  the  table  on  page  304. 


304  THE   BLOOD 

VARIETIES   OF   THE   MALARIAL   ORGANISM 


Tkrtian. 

Quartan. 

ESTIVO-AUTUMNAL. 

Asexual  cycle,  forty-eight 
hours. 

.  Seventy-two  hours. 

Usually  twenty-four  to 
forty-eight  hours. 

Substance  pale,  trans- 
parent,   comparable   to 
hyaline  tube-cast. 

Outline  indistinct. 

Ameboid  motion  ac- 
tive. 

Mature  asexual  form 
large;  fills  and  often  dis- 
tends corpuscle. 

Pigment -granu'  -s 
fine,     brown,    scattered 
throughout.      Very   ac- 
tive dancing  motion. 

Segmenting  body 
rarely   assumes   typical 
"daisy"   form.      15    to 
JO  segments. 

Gametes  resemble 
asexual  forms. 

Red   corpuscles  pale 
and  swollen. 

Highly     refractive, 
comparable     to     waxy 
lube-cast. 

Distinct. 

Sluggish. 

Smaller. 

Much  coarser,  darker 
in  color,  peripherally  ar- 
ranged.   Motion  slight. 

Usually    tyjiical 
"  daisy."     6  to  12  seg- 
ments. 

Same  as  tertian. 

Generally  darker  than 
normal. 

Highly  refractive. 

Distinct. 
Active. 

Young  forms,  only, 
in  peripheral  blood. 

Very    few,    minute, 
inactive.         Distinctly 
pigmented    forms   sel- 
dom seen. 

Not    seen    in    peri- 
pheral blood. 

Appear  in  blood  as 
distinctive  ovoids  and 
crescents. 

Dark,  often  bronzed. 

4.  Filarial  Larvae. — A  description  of  the  filariae 
whose   larvae   appear   in   the   blood  will   be  found    on 

P-  356. 

The  embryos  can  be  seen  in  stained  preparations  (Fig. 
103),  but  are  best  found  in  fresh  unstained  blood.  A 
rather  large  drop  is  taken  upon  a  slide,  covered,  and  ex- 
amined with  a  low  power.  The  embr}^©  can  be  located 
by  the  commotion  which  its  active  motion  produces 


BLOOD   PARASITES  305 

among  the  corpuscles.  This  motion  consists  almost 
wholly  in  apparently  purposeless  lashing  and  coiling 
movements,  and  continues  for  many  hours. 

5.  Larvae  of  Trichinella  spiralis. — The  worm  and 
its  life-history  are  described  on  page  414.  In  1909 
Herrick  and  Janeway  demonstrated  that  diagnosis  of 
trichiniasis  can  frequently  be  made  by  detection  of  the 
larvae  in  the  blood  during  their  migration  to  the 
muscles.    Of  the  examinations  which  have  been  reported 


Fig.  103. — Filarial  larvae  in  blood.    Stained.    Red  corpuscles  decolorized;  a  few  leuko- 
cytes remain  (X2cx>)  (photographs  by  the  author). 


since  that  time,  about  one-half  have  been  positive.  The 
earliest  time  at  which  the  embryos  were  found  was  the 
sixth  day  after  the  onset  of  symptoms;  the  latest,  the 
twenty-second  day. 

The  method  is  very  simple.  One  to  10  c.c.  of  blood 
are  obtained  from  the  ear  or  a  vein,  as  described  on  page 
293,  and  mixed  with  ten  times  its  volume  of  3  per  cent, 
acetic  acid.  The  mixture  is  centrifugalized,  .and  large 
drops  of  the  sediment  are  placed  on  slides,  covered,  and 
searched  with  a  low-power  objective.  The  larvae  are 
20 


306  THE   BLOOD 

not  difficult  to  recognize.    They  are  about  12$  fi  long 
and  6  (J.  broad. 

X.  TESTS  FOR  RECOGNITION  OF  BLOOD 
The  recognition  of  red  blood-corpuscles  microscopic- 
ally is  the  surest  and  simplest  means  of  detecting  the 
presence  of  blood.  In  most  pathologic  material,  how- 
ever, the  corpuscles  are  too  much  degenerated  for  recog- 
nition with  the  microscope,  and  one  has  to  rely  upon  a 
test  for  hemoglobin  or  its  derivatives.  Of  such  tests, 
the  two  given  in  this  section  are  probably  the  best. 
Each  is  reliable  within  its  own  sphere,  but  each  has  its 
limitation.  The  guaiac  test  is  reliable  only  when  nega- 
tive. The  same  is  true  of  its  modifications,  such  as  the 
benzidin  test.  The  hemin  test  is  reUable  only  when 
positive.  When,  however,  proper  care  is  taken  to  ex- 
clude fallacies,  the  guaiac  test  and  its  modifications 
are  the  most  useful  and  reliable  tests  for  clinical  pur- 
poses, although  they  could  not  be  accepted  medico- 
legally. 

The  only  reliable  test  for  human  blood  as  distinguished 
from  that  of  animals  is  the  precipitin  test  described  on 

P-  503- 

I.  Guaiac  Test.— The  technic  of  this  test  has  been 
given  (see  p.  157).  It  may  be  applied  directly  to  a  sus- 
pected fluid  or,  better,  to  the  ethereal  extract.  Add  a  few 
cubic  centimeters  of  glacial  acetic  acid  to  about  10  c.c. 
of  the  fluid;  shake  thoroughly  with  an  equal  volume  of 
ether;  decant,  and  apply  the  test  to  the  ether.  Jager 
states  that  the  test  is  rendered  much  more  sensitive  if 
a  few  drops  of  ammonia  or  sodium  hydroxid  solution 
be  added  to  the  ether  extract.    In  case  of  dried  stains 


TESTS  FOR  RECOGNITION  OF  BLOOD      307 

upon  cloth,  wood,  etc.,  dissolve  the  stain  in  distilled 
water  and  test  the  water,  or  press  a  piece  of  moist 
blotting-paper  against  the  stain,  and  touch  the  paper 
with  drops  of  the  guaiac  and  the  turpentine  successively. 
The  test  may  be  applied  to  microscopic  particles  by 
running  the  reagents  under  the  cover-glass. 

The  benzidin  test  (see  p.  157)  is  similar  to  the  guaiac 
test  and  has  the  same  fallacies,  but  is  said  to  be  more 
sensitive. 


Fig.  104. — Teichmann's  hemin  crystals  (Jakob). 

2.  Teichmann's  Test. — This  depends  upon  the  pro- 
duction of  characteristic  crystals  of  hemin.  It  is  a  sensi- 
tive test  and,  when  positive,  is  absolute  proof  of  the 
presence  of  blood.  A  number  of  substances — lime,  fine 
sand,  iron  rust — interfere  with  production  of  the  crys- 
tals; hence  negative  results  are  not  always  conclusive. 
Dissolve  the  suspected  stain  in  a  few  drops  of  normal 
salt  solution  upon  a  slide.  If  a  liquid  is  to  be  tested, 
evaporate  some  of  it  upon  a  slide  and  dissolve  the  residue 
in  a  few  drops  of  the  salt  solution.     Let  dry,  apply  a 


3o8  THE   BLOOD 

cover-glass,  and  run  glacial  acetic  acid  underneath  it. 
Heat  very  gently  until  bubbles  begin  to  form,  replacing 
the  acid  as  it  evaporates.  Allow  to  cool  slowly.  When 
cool,  replace  the  acid  with  water,  and  examine  for 
hemin  crystals  with  i6-nmi.  and  4-mm.  objectives.  The 
crystals  are  dark-brown  rhombic  plates,  lying  singly 
or  in  crosses,  and  easily  recognized  (Fig.  104).  Failure 
to  obtain  them  may  be  due  to  too  much  salt,  too  great 
heat,  or  too  rapid  cooling.  If  not  obtained  at  first,  let 
the  slide  stand  in  a  warm  place,  as  upon  a  hot-water 
radiator,  for  an  hour. 

XI.  SPECIAL  BLOOD  PATHOLOGY 

The  more  conspicuous  characteristics  of  the  blood  in 
various  diseases  have  been  mentioned  in  previous  sec- 
tions. Although  the  great  majority  of  blood  changes  are 
secondary,  there  are  a  few  blood  conditions  in  which  the 
changes  are  so  prominent,  or  the  etiology  so  obscure, 
that  they  are  commonly  regarded  as  blood  diseases. 
These  will  receive  brief  consideration  here.  They  fall 
into  two  general  groups.  In  the  one  group  (Anemia) 
the  red  cells  and  hemoglobin  are  chiefly  affected;  in  the 
other  (Leukemia)  changes  in  the  leukocytes  constitute 
the  conspicuous  feature  of  the  blood-picture. 

A.    Anemia 

This  is  a  deficiency  of  hemoglobin  or  red  corpuscles, 
or  both.  It  is  either  primary  or  secondary.  The  dis- 
tinction is  based  chiefly  upon  etiology,  although  each 
type  presents  a  more  or  less  distinctive  blood-picture. 
Secondary  anemia  is  that  which  is  symptomatic  of  some 


SPECIAL   BLOOD   PATHOLOGY  309 

other  pathologic  condition.  Primary  anemia  is  that 
which  progresses  without  apparent  cause. 

1.  Secondary  Anemia. — The  more  important  condi- 
tions which  produce  secondary  or  symptomatic  anemia 
are: 

(a)  Poor  nutrition,  which  usually  accompanies  un- 
sanitary conditions,  poor  and  insufficient  food,  etc. 

(6)  Acute  infectious  diseases,  especially  rheumatism 
and  typhoid  fever.  The  anemia  is  more  conspicuous 
during  convalescence. 

(c)  Chronic  Infectious  Diseases. — Tuberculosis,  syph- 
ilis, leprosy. 

{d)  Chronic  exhausting  diseases,  as  heart  disease, 
chronic  nephritis,  cirrhosis  of  the  Uver,  and  gastro- 
intestinal diseases,  especially  when  associated  with 
atrophy  of  gastric  and  duodenal  glands.  The  last  may 
give  an  extreme  anemia,  indistinguishable  from  perni- 
cious anemia. 

(e)  Chronic  poisoning,  as  from  lead,  arsenic,  and 
phosphorus. 

(/)  Hemorrhage. — EitTier  repeated  small  hemorrhages 
(chronic  hemorrhage),  as  from  gastric  cancer  and  ulcer, 
hemorrhoids,  uterine  fibroids,  etc.,  or  acute  hemor- 
rhage, such  as  may  occur  in  typhoid  fever,  tuberculo- 
sis, or  traumatism. 

{g)  Malignant  Tumors. — These  affect  the  blood  partly 
through  repeated  small  hemorrhages,  partly  through 
toxic  products,  and  partly  through  interference  with 
nutrition. 

Qi)  Animal  Parasites. — Some  cause  no  appreciable 
change  in  the  blood;  others,  like  the  hookworm  and 
Dibothriocephalus   latus,   may   produce   a   very   severe 


310  THE    BLOOD 

anemia,  almost  identical  with  pernicious  anemia.  Ane- 
mia in  these  cases  is  probably  due  both  to  toxins  and  to 
abstraction  of  blood.  In  malaria  the  parasites  them- 
selves directly  destroy  the  red  cells. 

The  blood-picture  varies  with  the  grade  of  anemia. 
Diminution  of  hemoglobin  is  the  most  characteristic 
feature.  In  mild  cases  it  is  slight,  and  is  the  only  blood 
change  to  be  noted.  In  very  severe  cases  hemoglobin 
may  fall  to  15  per  cent,  or  even  lower.  Red  corpuscles 
are  diminished  in  all  but  very  mild  cases,  while  in  the 
severest  cases  the  red  corpuscle  count  is  sometimes 
below  2,000,000.    The  color-index  is  usually  decreased. 

Although  the  number  of  leukocytes  bears  no  relation 
to  the  anemia,  leukocytosis  is  common,  being  due  to  the 
same  cause. 

Stained  films  show  no  changes  in  very  mild  cases.  In 
moderate  cases  variations  in  size  and  shape  of  the  red 
cells  and  polychromatophilia  occur.  Very  severe  cases 
show  the  same  changes  to  greater  degree,  with  addition 
of  basophilic  degeneration  and  the  presence  of  normo- 
blasts in  small  or  moderate  numbers.  Megaloblasts 
in  very  small  numbers  have  been  encountered  in  ex- 
tremely severe  cases.  They  are  especially  abundant 
and  may  even  predominate  over  the  normoblasts  in 
dibothriocephalus  infection.  Blood-plaques  are  usually 
increased. 

Posthemorrhagic  Anemia. — Within  a  few  hours  after 
an  acute  hemorrhage  the  volume  of  blood  is  nearly  or 
quite  restored  by  means  of  fluids  from  the  tissues. 
Owing  to  the  fact  that  some  destruction  of  red  corpus- 
cles continues  for  a  time,  the  anemia  is  most  marked 
a  few  days  after  the  hemorrhage.    Hemoglobin  and  red 


SPECIAL    BLOOD   PATHOLOGY  31I 

cells  are  diminished  according  to  the  amount  of  blood 
lost.  The  color-index  is  moderately  low.  There  is 
moderate  leukocytosis.  Some  of  the  red  cells  may  show 
polychromatophilia  and  a  few  normoblasts  may  be 
found.  In  some  cases  great  numbers  of  normoblasts 
appear  rather  suddenly— a  so-called  blood  crisis.  Nor- 
mal conditions  may  be  restored  within  a  few  weeks, 
although  the  color-index  is  apt  to  remain  low  for  some 
time  thereafter. 

2.  Primary  Anemia.— The  commonly  described  vari- 
eties of  primary  anemia  are  pernicious  anemia  and  chlo- 
rosis, but  splenic  anemia  may  also  be  mentioned  under 
this  head. 

(i)  Progressive  Pernicious  Anemia. — It  is  frequently 
impossible  to  diagnose  this  disease  from  the  blood  ex- 
amination alone.  Severe  secondary  anemia,  especially 
that  due  to  gastro-intestinal  cancer,  intestinal  parasites, 
and  repeated  small  hemorrhages,  sometimes  gives  an 
identical  picture.  Remissions,  in  which  the  blood 
approaches  the  normal,  are  common.  All  the  clinical 
data  must,  therefore,  be  considered,  together  with  a 
careful  analysis  of  repeated  blood  examinations. 

The  disease  is  characterized  by  active  destruction  of 
red  corpuscles  with  excessive  activity  of  the  erythro- 
blastic bone-marrow,  and  the  appearance  of  immature 
and  abnormal  red  cells  in  the  circulation. 

Hemoglobin  and  red  corpuscles  are  always  greatly 
diminished.  Several  counts  in  which  the  red  cells 
were  below  150,000  have  been  recorded.  In  none  of 
.Cabot's  139  cases  did  the  count  exceed  2,500,000,  the 
average  being  about  1,200,000.  In  more  than  two- 
thirds  of   the   cases  hemoglobin  was  reduced    to  less 


312 


THE   BLOOD 


extent  than  the  red  corpuscles;  the  color-index  was, 
therefore,  high.  A  low  color-index  probably  indicates 
a  mild  type  of  the  disease.  The  average  hemoglobin 
value  is  about  25  to  30  per  cent. 

The  leukocyte  count  may  be  normal,  but  is  commonly 
diminished  to  about  3000,  and  is  sometimes  much  lower. 


Fig.  105. — A,  Normal  blood;  B,  chlorosis;  C,  pernicious  anemia.  The  plate  shows 
the  sharp  contrast  between  cells  rich  in  hemoglobin  and  the  pale  cells  of  chlorosis,  and  also 
the  poikilocytes  and  marked  variations  in  size  noted  in  pernicious  anemia.  A  normo- 
blast and  megaloblast  also  appear.    Stained  smears  {from  Greene's  "Medical  Diagnosis"). 

The  decrease  affects  chiefly  the  polymorphonuclear  cells, 
so  that  the  l^Tnphocytes  are  relatively  increased.  In 
some  cases  a  decided  absolute  increase  of  lymphocytes 
occurs.  Polymorphonuclear  leukocytosis,  when  present, 
is  due  to  some  complication. 

The  red  corpuscles  show  marked  variation  in  size  and 


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Explanation  of  Plate  IX 

Pig.  I. — Preparation  from  an  advanced  case  of  progressive  perni- 
cious anemia  from  unknown  cause:  a,  Megaloblasts or  gigantoblasts;  the 
protoplasm  shows  marked  polychromasia;  b,  stained  granules  in  ervthro- 
cytes  with  normally  stained  protoplasm;  c  and  d,  polychromatophilic 
degeneration;  e,  megalocytes;  /,  normocytes. 

Fig.  2. — Preparation  from  the  same  case  taken  some  time  later 
while  the  patient  was  subjectively  and  objectively  in  perfect  health: 
a,  Punctate  erj-throcytes  with  normal  and  anemic  degenerated  proto- 
plasm; b,  polynudear  leukocyte;  c,  normal  red  blood-corpuscles;  d, 
somewhat  enlarged  erythrocytes. 

Pig.  3. — Series  of  cells  from  a  case  of  severe  progressive  per- 
nicious anemia  of  unknown  etiology;  preparation  made  two  days  ante- 
mortem:  a,  Nucleated  red  blood-corpuscles  characterized  as  normo- 
blasts by  the  intense  staining  of  the  nuclei;  a'  and  a",  karyokinetic 
figures  in  erythrocytes;  the  protoplasm  finely  punctate;  b,  beginning 
karyolysis  in  a  megaloblast;  c,  erythroblasts  with  coarse  granulation  of 
the  protoplasm;  d,  nuclear  remains  (?)  and  fine  granulation  of  the 
protoplasm;  e  and  /,  finely  punctate  red  blood-corpuscles;  g,  megalocyte 
with  two  blue  nuclei;  nuclear  remains  (?)  in  the  polychrome  protoplasm. 

( Nothnagel  -Lazarus. ) 


.eu  pr 


PLATE  IX 


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SPECIAL   BLOOD   PATHOLOGY  313 

shape  (Plate  IX  and  Fig.  105).  There  is  a  decided 
tendency  to  large  oval  forms,  and,  despite  the  presence 
of  microcytes,  the  average  size  of  the  corpuscles  is  gen- 
erally strikingly  increased.  Polychromatophilia  and 
basophilic  degeneration  are  common.  Nucleated  red 
cells  are  always  present,  although  in  many  instances  care- 
ful search  is  required  to  find  them.  In  the  great  majority 
of  cases  megaloblasts  exceed  normoblasts  in  number. 
This  ratio  is  practically  unknown  in  other  diseases. 
Blood-plaques  are  diminished. 

The  chief  points  to  be  considered  in  diagnosis  are  the 
high  color-index  and  the  presence  of  megaloblasts. 

The  rare  and  rapidly  fatal  anemia  which  has  been 
described  under  the  name  of  aplastic  anemia  is  probably 
a  variety  of  pernicious  anemia.  Absence  of  any  attempt 
at  blood  regeneration  explains  the  marked  difference  in 
the  blood-picture.  Red  corpuscles  and  hemoglobin  are 
rapidly  diminished  to  an  extreme  degree.  The  color- 
index  is  normal  or  low.  The  leukocyte  count  is  normal 
or  low,  with  relative  increase  of  lymphocytes.  Stained 
smears  show  only  slight  variations  in  size,  shape,  and 
staining  properties  of  the  red  cells.  There  are  no  megalo- 
blasts and  few  or  no  normoblasts. 

(2)  Chlorosis. — This  is  probably  a  disease  of  defective 
blood  formation.  It  is  confined  almost  exclusively  to 
unmarried  girls.  The  clinical  symptoms  furnish  the 
most  important  data  for  diagnosis.  The  blood  resembles 
that  of  secondary  anemia  in  many  respects. 

The  most  conspicuous  feature  is  a  marked  decrease  of 
hemoglobin,  accompanied  by  a  slight  decrease  in  number 
of  red  corpuscles.  The  color-index  is  thus  almost  in- 
variably low. 


314  THE    BLOOD 

The  following  figures  represent  about  the  average  for 
well-marked  cases:  hemoglobin,  40  per  cent.;  red  cor- 
puscles, 4,000,000;  color-index,  0.5.  Much  lower  figures 
are  frequent;  while,  upon  the  other  hand,  mild  cases  may 
show  no  loss  at  all  in  number  of  red  cells. 

As  in  pernicious  anemia,  the  leukocytes  are  normal  or 
decreased  in  number,  with  a  relative  increase  of  lym- 
phocytes. 

In  contrast  to  pernicious  anemia  (and  in  some  degree 
also  to  secondary  anemia),  the  red  cells  are  of  nearly 
uniform  size,  are  pale  (see  Fig.  105),  and  their  average 
diameter  is  somewhat  less  than  normal.  Changes  in 
size,  shape,  and  staining  reactions  occur  only  in  severe 
cases.  Erythroblasts  are  rarely  present.  The  number 
of  plaques  is  generally  decreased. 

(3)  Splenic  Anemia. — This  is  an  obscure  form  of 
anemia  associated  with  great  enlargement  of  the  spleen. 
It  is  probably  a  distinct  entity,  although  several  types 
may  exist.  There  is  decided  decrease  of  hemoglobin 
and  red  corpuscles,  with  moderate  leukopenia  and  rela- 
tive lymphocytosis.  Osier's  15  cases  averaged  47  per 
cent,  hemoglobin  and  3,336,357  red  cells.  Stained  fihns 
show  notable  irregularities  in  size,  shape,  and  staining 
properties  only  in  advanced  cases.  Erythroblasts  are 
uncommon. 

B.    Leukemia 

Except  in  rare  instances,  diagnosis  is  easily  made 
from  the  blood  alone,  usually  at  the  first  glance  at  the 
stained  film.  Two  types  of  the  disease  are  commonly 
distinguished:  the  myelogenous  and  the  lymphatic. 
Atypical    forms   are   not   uncommon.     The   disease   is 


SPECIAL   BLOOD   PATHOLOGY  315 

characterized  by  hyperplasia  of  the  leukoblastic  bone- 
marrow  (myelogenous  leukemia)  or  of  the  lymphoid 
tissues  (lymphatic  leukemia),  together  with  overflow 
of  many  immature  leukocytes  and  excessive  numbers 
of  normal  types  into  the  circulating  blood.  The  more 
acute  the  process,  the  more  immature  are  the  cells  which 
appear  in  the  blood. 

1,  Myelogenous  Leukemia  (Plate  X). — This  is  usu- 
ally a  chronic  disease,  although  acute  cases  have  been 
described. 

Hemoglobin  and  red  corpuscles  show  decided  decrease. 
The  red  count  is  usually  below  3,500,000.  Accurate 
hemoglobin  estimation  is  difficult  because  of  the  great 
number  of  leukocytes.  The  color-index  is  moderately 
low. 

Most  striking  is  the  immense  increase  in  number  of 
leukocytes.  The  count  in  ordinary  cases  varies  between 
100,000  and  400,000.  Counts  over  1,000,000  have  been 
met.  During  spontaneous  remissions,  during  treatment 
with  a;-ray  or  benzol,  and  during  intercurrent  infections 
the  leukocyte  count  may  fall  to  normal. 

While  these  enormous  leukocyte  counts  are  equaled  in 
no  other  disease,  and  approached  only  in  lymphatic 
leukemia  and  extremely  high-grade  leukocytosis,  the 
diagnosis,  particularly  during  remissions,  depends  more 
upon  quaHtative  than  quantitative  changes.  Although 
all  varieties  are  increased,  the  characteristic  and  con- 
spicuous cell  is  the  myelocyte.  This  cell  never  appears 
in  normal  blood;  extremely  rarely  in  leukocytosis;  and 
jiever  abundantly  in  l)rmphatic  leukemia.  In  myelog- 
enous leukemia  myelocytes  usually  constitute  more  than 
20  per  cent,  of  all  leukocytes.     Da  Costa's  lowest  case 


3l6  THE   BLOOD 

gave  7  per  cent.  The  neutrophilic  form  is  generally 
much  more  abundant  than  the  eosinophilic.  Both  show 
considerable  variations  in  size.  Myeloblasts  may  be 
present  in  small  numbers  at  any  stage,  and  in  the  ter- 
minal stages  they  may  be  abundant.  An  increase  in 
their  number  is  of  grave  significance.  Very  constant  in 
myelogenous  leukemia  is  a  marked  absolute,  and  often 
a  relative,  increase  of  eosinophiles  and  basophiles.  Poly- 
morphonuclear neutrophilcs  and  lymphocytes  are  abso- 
lutely increased,  although  relatively  decreased. 

The  red  cells  show  the  changes  characteristic  of  a 
severe  secondary  anemia,  except  that  nucleated  reds  are 
commonly  abundant;  in  fact,  no  other  disease  gives  so 
many.  They  are  chiefly  of  the  normoblastic  type.  Meg- 
aloblasts  are  uncommon.  Blood-plaques  are  generally 
increased. 

In  acute  myelogenous  leukemia  the  myeloblast  may 
be  the  predominant  cell,  and  the  blood  will  then  re- 
semble that  of  acute  lymphatic  leukemia.  The  myelo- 
blast can  be  distinguished  from  the  large  lymphocyte 
by  the  oxydase  reaction  (see  p.  290)  although  cases 
occur  in  which  the  t3^e  of  blood  formation  is  so  em- 
bryonic that  the  oxydase  reaction  fails. 

2.  Lymphatic  Leukemia  (Plate  X). — Chronic  Form. 
— There  is  generally  greater  loss  of  hemoglobin  and  red 
corpuscles  than  in  myelogenous  leukemia.  The  color- 
index  is  usually  moderately  low. 

The  leukocyte  count  is  high,  but  lower  than  in  the 
myelogenous  type.  Counts  of  100,000  to  200,000  are 
about  the  average,  but  in  many  cases  are  much  lower. 
This  high  count  is  referable  almost  wholly  to  increase 
of  lymphocytes.     They  generally  exceed  90  per  cent. 


PLATE  X 


Fig.  I. — Blood  in  lymphatic  leukemia;  X  700.     On  the  left,  chronic  form 
of  the  disease;  on  the  right,  acute  form  (courtesy  of  Dr.  W.  P.  Harlow). 


Fi^'  2. — Blood  in  splenomyelogenous  leukemia.     Wright's  stain.     X  700 
(photographs  by  the  author). 


SPECIAL   BLOOD   PATHOLOGY  317 

of  the  total  number  and  are  chiefly  of  the  small  variety. 
During  remissions  the  total  count  may  fall  to  below 
normal,  but  the  percentage  of  lymphocytes  remains 
high.     Myelocytes  are  rare. 

The  red  corpuscles  show  the  changes  usual  in  severe 
secondary  anemia.  Erythroblasts  are  seldom  abundant. 
Blood-plaques  are  decreased. 

Acute  Form. — The  blood  is  similar  to  that  of  the 
chronic  variety.  The  total  leukocyte  count  is  seldom 
so  high,  and  the  large  type  of  lymphocyte  predominates 
in  most  cases.  The  anemia  is  apt  to  be  more  severe 
and  the  normoblasts  more  abundant. 

3.  Anaemia  Infantum  Pseudoleukaemica. — Under 
this  name  von  Jaksch  described  a  rare  disease  of  infancy, 
the  proper  classification  of  which  is  uncertain.  There  is 
enlargement  of  liver  and  spleen,  and  sometimes  of 
lymph-nodes,  together  with  the  following  blood  changes: 
grave  anemia  with  deformed  and  degenerated  red  cells 
and  many  erythroblasts  of  both  normoblastic  and  mega- 
loblastic types;  great  increase  in  number  of  leukocytes 
(20,000  to  100,000)  and  great  variations  in  size,  shape, 
and  staining  of  leukocytes,  with  many  atypic  forms,  and 
a  few  myelocytes. 

From  the  work  of  more  recent  investigators  it  appears 
probable  that  von  Jaksch 's  anemia  is  not  a  distinct  dis- 
ease, and  that  the  reported  cases  have  been  atypical 
forms  of  leukemia,  pernicious  anemia,  or  even  secondary 
anemia  with  leukocytosis.  As  is  well  known,  all  of  these 
conditions  are  apt  to  be  atypical  in  children. 
-  The  table  on  the  following  page  contrasts  the  distinct- 
ive blood-changes  in  the  more  common  conditions. 


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THE    BLOOD 


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CHAPTER  IV 

THE  STOMACH 

Laboratory  methods  may  be  applied  to  the  diagnosis 
of  stomach  disorders  in:  I.  Examination  of  the  gastric 
contents  removed  with  the  stomach- tube.  II.  Certain 
other  examinations  which  give  information  as  to  the 
condition  of  the  stomach. 

I.  EXAMINATION  OF  THE  GASTRIC  CONTENTS 

Stomach  digestion  consists  mainly  in  the  action  of  pep- 
sin upon  proteins  in  the  presence  of  hydrochloric  acid 
and  in  the  curdling  of  milk  by  rennin.  The  fat-splitting 
ferment,  lipase,  of  the  gastric  juice  has  very  little  activity 
under  normal  conditions  of  acidity. 

Pepsin  and  rennin  are  secreted  by  the  gastric  glands 
as  zymogens — pepsinogen  and  renninogen  respectively 
—which  are  converted  into  pepsin  and  rennin  by  hydro- 
chloric acid.  Hydrochloric  acid  is  secreted  by  certain 
cells  of  the  fundus  glands.  It  at  once  combines  loosely 
with  the  proteins  of  the  food,  forming  acid-metaprotein, 
the  first  step  in  protein  digestion.  Hydrochloric  acid, 
which  is  thus  loosely  combined  with  proteins,  is  called 
"combined"  hydrochloric  acid.  The  acid  which  is  se- 
creted after  the  proteins  present  have  all  been  converted 
into  acid-metaprotein  remains  as  "free"  hydrochloric 
acid,  and,  together  with  pepsin,  continues  the  process  of 
digestion. 

319 


320  THE   STOMACH 

At  the  height  of  digestion  the  stomach-contents  con- 
sist essentiallly  of:  (i)  Water;  (2)  free  hydrochloric  acid; 
(3)  combined  hydrochloric  acid;  (4)  pepsin;  (5)  rennin; 
(6)  mineral  salts,  chiefly  acid  phosphates,  of  no  clinical 
importance;  (7)  particles  of  undigested  and  partly  di- 
gested food;  (8)  various  products  of  digestion  in  solution. 
In  pathologic  conditions  there  may  be  present,  in  addi- 
tion, various  microscopic  structures  and  certain  organic 
acids,  of  which  lactic  acid  is  most  important. 

A  routine  examination  is  conveniently  carried  out  in  the 
following  order: 

(i)  Give  the  patient  a  test-meal  upon  an  empty  stomach, 
washing  the  stomach  previously  if  necessary. 

(2)  At  the  height  of  digestion,  usually  in  one  hour,  remove 
the  contents  of  the  stomach  with  a  stomach-tube. 

(3)  Measure  and  examine  macroscopically. 

(4)  Filter.  A  suction  filter  is  desirable,  and  may  be  neces- 
sary when  much  mucus  is  present. 

(5)  During  filtration,  examine  microscopically  and  make 
qualitative  tests  for — (a)  free  acids;  (6)  free  hydrochloric 
acid;  (c)  lactic  acid. 

(6)  When  sufficient  filtrate  is  obtained,  make  quantitative 
estimations  of — (a)  total  acidity;  (b)  free  hydrochloric  acid; 
(c)  combined  hydrochloric  acid  (if  necessary) . 

(7)  Make  whatever  additional  tests  seem  desirable,  as  for 
blood,  pepsin,  or  rennin. 

A.   Obtaining  the  Contents 

Gastric  juice  is  secreted  continuously,  but  quantities 
sufficiently  large  for  examination  are  not  usually  obtain- 
able from  the  fasting  stomach.  In  clinical  work,  there- 
fore, it  is  desirable  to  stimulate  secretion  with  food — 


EXAMINATION   OF  THE   GASTRIC  CONTENTS        32 1 

which  is  the  natural  and  most  efficient  stimulus — before 
attempting  to  collect  the  gastric  fluid.  Diflferent  foods 
stimulate  secretion  to  different  degrees,  hence  for  the 
sake  of  uniform  results  certain  standard  "test-meals" 
have  been  adopted. 

1 .  Test=meals. — It  is  customary  to  give  the  test-meal 
in  the  morning,  since  the  stomach  is  most  apt  to  be 
empty  at  that  time.  If  it  be  suspected  that  the  stomach 
will  not  be  empty,  it  should  be  washed  out  with  water 
the  evening  before. 

(i)  Ewald's  test-breakfast  consists  of  a  roll  (or  two 
slices  of  bread) ,  without  butter,  and  two  small  cups  (300 
to  400  c.c.)  of  water,  or  weak  tea,  without  cream  or 
sugar.  It  should  be  well  masticated.  The  contents  of  the 
stomach  are  to  be  removed  one  hour  afterward,  counting 
from  the  beginning,  not  the  end,  of  the  meal.  This 
test-meal  has  long  been  used  for  routine  examinations. 
Its  disadvantage  is  that  it  introduces,  with  the  bread,  a 
variable  amount  of  lactic  acid  and  numerous  yeast-cells. 
This  source  of  error  may  be  eliminated  by  substituting 
a  shredded  whole-wheat  biscuit  for  the  roll.  The 
shredded  wheat  test-meal  is  now  widely  used  and  is 
probably  the  most  satisfactory  for  general  purposes. 

(2)  Boas'  test-breakfast  consists  of  a  tablespoonful 
of  rolled  oats  in  a  quart  of  water,  boiled  to  one  pint,  with 
a  pinch  of  salt  added.  It  should  be  withdrawn  in  forty- 
five  minutes  to  one  hour.  This  meal  does  not  contain 
lactic  acid,  and  is  usually  given  when  the  detection  of 
lactic  acid  is  important,  as  in  suspected  gastric  cancer. 
The  stomach  should  always  be  washed  with  water  the 
evening  previous. 

(3)  Riegel's  test-meal  consists  of  400  c.c.  of  bouillon, 

21 


322  THE    STOMACH 

a  broiled  beefsteak  (about  150-200  gm.),  and  150  gm.  of 
mashed  potato.  Since  it  tends  to  clog  the  tube,  it  must 
be  thoroughly  masticated. 

(4)  Fischer's  test-meal  is  similar,  but  probably  pref- 
erable. It  consists  of  an  Ewald  breakfast  plus  about 
J  pound  lean,  finely  chopped  Hamburger  steak,  broiled, 
and  lightly  seasoned.  This  and  Riegel's  may  be  re- 
moved in  three  to  four  hours.  They  give  somewhat 
higher  acidity  values  than  the  Ewald  breakfast. 

2.  Withdrawal  of  the  Contents.— The  Boas  stomach- 
tube,  with  bulb,  is  probably  the  most  satisfactory  form. 
It  should  be  of  rather  large  caliber,  and  have  an  opening 
in  the  tip  and  one  or  two  in  the  side  near  the  tip.  When 
not  in  use  it  should  be  kept  in  a  vesse  of  borax  solution, 
and  should  be  well  washed  in  hot  water  both  before  and 
after  using. 

It  is  important  confidently  to  assure  the  patient  that 
introduction  of  the  tube  cannot  possibly  harm  him;  and 
that,  if  he  can  control  the  spasm  of  his  throat,  he  will 
experience  very  little  choking  sensation.  When  patients 
are  very  nervous  it  is  well  to  spray  the  throat  with  co- 
cain  solution. 

The  tube  should  be  dipped  in  warm  water  just  before 
using;  the  use  of  glycerin  or  other  lubricant  is  undesir- 
able. With  the  patient  seated  upon  a  chair,  his  cloth- 
ing protected  by  towels  or  a  large  apron,  and  his  head 
tilted  forward,  the  tip  of  the  tube,  held  as  one  would  a 
pen,  is  introduced  far  back  into  the  pharynx.  He  is  then 
urged  to  swallow,  and  the  tube  is  pushed  boldly  into  the 
esophagus  until  the  ring  upon  it  reaches  the  incisor  teeth, 
thus  indicating  that  the  tip  is  in  the  stomach.  If,  now, 
the  patient  cough  or  strain  as  if  at  stool,  the  contents  of 


EXAMINATION    OF  THE    GASTRIC   CONTENTS         323 

the  stomach  will  usually  be  forced  out  through  the  tube. 
Should  it  fail,  the  fluid  can  generally  be  pumped  out  by 
alternate  compression  of  the  tube  and  the  bulb.  If 
unsuccessful  at  first,  the  attempts  should  be  repeated 
with  the  tube  pushed  a  little  further  in,  or  withdrawn  a 
few  inches,  since  the  distance  to  the  stomach  is  not  the 
same  in  all  cases.  The  tube  may  become  clogged  with 
pieces  of  food,  in  which  case  it  must  be  withdrawn, 
cleaned,  and  reintroduced.  If,  after  all  efforts,  no  fluid 
is  obtained,  another  test-meal  should  be  given  and  with- 
drawn after  a  somewhat  shorter  period,  since,  owing 
to  excessive  motility,  the  stomach  may  empty  itself  in 
less  than  the  usual  time. 

Care  must  be  exercised  to  prevent  saliva  running  down 
the  outside  of  the  tube  and  mingling  with  the  gastric  juice 
in  the  basin. 

As  the  tube  is  removed,  it  should  be  pinched  between 
the  fingers  so  as  to  save  any  fluid  that  may  be  in  it. 

The  stomach-tube  must  be  used  with  great  care,  or  not 
at  all,  in  cases  of  gastric  ulcer,  aneurysm,  uncompensated 
heart  disease,  and  marked  arteriosclerosis.  Except  in 
gastric  ulcer,  the  danger  lies  in  the  retching  produced,  and 
the  tube  can  safely  be  used  if  the  patient  takes  it  easily. 

B.  Physical  Examination 
Under  normal  conditions  30  to  50  c.c.  of  fluid  can  be 
obtained  one  hour  after  administering  Ewald's  break- 
fast. More  than  60  c.c.  points  to  motor  insufficiency; 
less  than  20  c.c,  to  too  rapid  emptying  of  the  stomach, 
or  else  to  incomplete  removal.  Upon  standing,  it  sepa- 
rates into  two  layers:  the  lower  consisting  of  particles  of 
food;  the  upper,  of  an  almost  clear,  faintly  yellow  fluid. 


324  THE  STOMACH 

The  extent  to  which  digestion  has  taken  place  can  be 
roughly  judged  from  the  appearance  of  the  food-particles. 

The  reaction  is  frankly  acid  in  health  and  in  nearly 
all  pathologic  conditions.  It  may  be  neutral  or  slightly 
alkaUne  in  some  cases  of  gastric  cancer  and  marked 
chronic  gastritis,  or  when  contaminated  by  a  consider- 
able amount  of  saliva. 

A  small  amount  of  mucus  is  present  normally.  Large 
amounts,  when  the  gastric  contents  are  obtained  with 
the  tube  and  not  vomited,  point  to  chronic  gastritis. 
Mucus  is  recognized  from  its  characteristic  slimy  appear- 
ance when  the  fluid  is  poured  from  one  vessel  into  another. 
It  is  more  frequently  seen  in  stomach  washings  than  in 
the  fluid  removed  after  a  test-meal. 

A  trace  of  bile  may  be  present  as  a  result  of  excessive 
straining  while  the  tube  is  in  the  stomach.  Large 
amounts  are  very  rarely  found,  and  generally  point  to 
obstruction  in  the  duodenum.  Bile  produces  a  yellowish 
or  greenish  discoloration  of  the  fluid. 

Blood  is  often  recognized  by  simple  inspection,  but 
more  frequently  requires  a  chemic  test.  It  is  bright  red 
when  very  fresh,  and  dark,  resembling  coffee-grounds, 
when  older.  Vomiting  of  blood,  or  hematemesis,  may  be 
mistaken  for  pulmonary  hemorrhage,  or  hemoptysis.  In 
the  former  the  fluid  is  acid  in  reaction  and  usually  dark 
red  or  brown  in  color  and  clotted,  while  in  hemoptysis 
it  is  brighter  red,  frothy,  alkaline,  and  usually  mixed 
with  a  variable  amount  of  mucus. 

Particles  of  food  eaten  hours  or  even  days  previously 
may  be  found,  and  indicate  deficient  motor  power. 

Search  should  always  be  made  for  bits  of  tissue  from 
the  gastric  mucous  membrane  or  new  growths.    These, 


EXAMINATION    OF   THE   GASTRIC   CONTENTS        325 

when  examined  by  a  pathologist,  will  sometimes  render 
the  diagnosis  clear. 

C.   Chemic  Examination 

A  routine  chemic  examination  of  the  gastric  contents 
involves  qualitative  tests  for  free  acids,  free  hydrochloric 
acid,  and  organic  acids,  and  quantitative  estimations  of 
total  acidity,  free  hydrochloric  acid,  and  sometimes 
combined  hydrochloric  acid.  Other  tests  are  applied 
when  indicated. 

1.  Qualitative  Tests.^(i)  Free  Acids.— The  pres- 
ence or  absence  of  free  acids,  without  reference  to  the 
kind,  is  easily  determined  by  means  of  Congo-red, 
although  the  test  is  not  much  used  in  practice. 

Congo-red  Test. — Take  a  few  drops  of  a  strong  alcoholic 
solution  of  Congo-red  in  a  test-tube,  dilute  with  water  to  a 
strong  red  color,  and  add  a  few  cubic  centimeters  of  filtered 
gastric  juice.  The  appearance  of  a  blue  color  shows  the 
presence  of  some  free  acid  (Plate  XI,  B,  B').  Since  the  test 
is  more  sensitive  to  mineral  than  to  organic  acids,  a  marked 
reaction  points  to  the  presence  of  free  hydrochloric  acid. 

Thick  filter-paper  soaked  in  Congo-red  solution,  dried,  and 
cut  into  strips  may  be  used,  but  the  test  is  much  less  delicate 
when  thus  applied. 

(2)  Free  Hydrochloric  Acid.^In  addition  to  its  diges- 
tive function,  free  hydrochloric  acid  is  an  efficient  anti- 
septic. It  prevents  or  retards  fermentation  and  lactic- 
acid  formation,  and  is  an  important  means  of  protection 
against  the  entrance  of  pathogenic  organisms  into  the 
body.    It  is  never  absent  in  health. 


326  THE    STOMACH 

Amidobenzol  Test. — To  a  little  of  the  filtered  gastric  juice 
in  a  test-tube,  or  to  several  drops  in  a  porcelain  dish,  add  a 
drop  of  0.5  per  cent,  alcoholic  solution  of  dimethylamido- 
azobenzol.  In  the  presence  of  free  hydrochloric  acid  there 
will  at  once  appear  a  cherry-red  color,  varying  in  intensity 
with  the  amount  of  acid  (Plate  XII,  C).  This  test  is  very 
delicate;  but,  unfortunately,  organic  acids,  when  present  in 
large  amounts  (above  0.5  per  cent.),  give  a  similar  reaction. 
The  color  obtained  with  organic  acids  is,  however,  more  of 
an  orange  red. 

Boas'  Test. — This  test  is  less  delicate  than  the  preceding, 
but  is  more  reliable,  since  it  reacts  only  to  free  hydrochloric 
acid.    It  is  probably  the  best  routine  test. 

In  a  procelain  dish  mix  a  few  drops  of  the  gastric  juice  and 
the  reagent,  and  slowly  evaporate  to  dryness  over  a  flame, 
taking  care  not  to  scorch.  The  appearance  of  a  rose-red  color, 
which  fades  upon  cooling,  shows  the  presence  of  free  hydro- 
chloric acid  (Plate  XI,  i). 

Boas^  reagent  consists  of  5  gm.  resublimed  resorcinol,  and 
3  gm.  cane-sugar,  in  100  c.c.  alcohol.  The  solution  keeps 
well,  which,  from  the  practitioner's  view-point,  makes  it 
preferable  to  Giinzburg's  phloroglucin-vanillin  reagent 
(phloroglucin,  2  gm.;  vanillin,  i  gm.;  absolute  alcohol, 
30  c.c).  The  latter  is  just  as  delicate,  is  appUed  in  the 
same  way,  and  gives  a  sharper  reaction  (Plate  XI,  2),  but 
is  unstable. 

(3)  Organic  Acids.— Lactic  acid  is  the  most  common, 
and  is  taken  as  the  type  of  the  organic  acids  which 
appear  in  the  stomach-contents.  It  is  a  product  of 
bacterial  acti\dty.  Acetic  and  butyric  acids  are  some- 
times present.  Their  formation  is  closely  connected 
with  that  of  lactic  acid,  and  they  are  rarely  tested  for. 
When  abundant,  they  may  be  recognized  by  their  odor 


PLATE  XI 


A' 


B' 


A,  Uffelmann's  reagent;  A',  A  after  the  addition  of  gastric  fluid 
containing  lactic  acid;  B,  water  to  which  three  drops  of  Congo-red 
solution  have  been  added;  B',  change  induced  in  B  when  gastric  fluid 
containing  free  hydrochloric  acid  is  added  (Boston). 


I,  Resorcin-test  for  free  hydrochloric  acid;  2,  Glinzburg's  test  for  hydro- 
chloric acid  (Boston). 


EXAMINATION    OF   THE   GASTRIC   CONTENTS        327 

upon  heating.  Butyric  acid  gives  the  odor  of  rancid 
butter. 

Lactic  acid  is  never  present  at  the  height  of  digestion 
in  health.  Although  usually  present  early  in  digestion, 
it  disappears  when  free  hydrochloric  acid  begins  to 
appear.  Small  amounts  may  be  introduced  with  the 
food.  Pathologically,  small  amounts  may  be  present 
whenever  there  is  stagnation  of  the  gastric  contents  with 
deficient  hydrochloric  acid,  as  in  many  cases  of  dilata- 
tion of  the  stomach  and  chronic  gastritis.  The  presence 
of  notable  amounts  of  lactic  acid  (more  than  o.i  per 
cent,  by  Strauss'  test)  is  strongly  suggestive  of  gastric 
cancer,  and  is  probably  the  most  valuable  single  symp- 
tom of  the  disease. 

As  already  stated,  the  Ewald  test-breakfast  introduces 
a  small  amount  of  lactic  acid,  but  rarely  enough  to  re- 
spond to  the  tests  given  here.  In  every  case,  however, 
in  which  its  detection  is  important,  the  shredded-wheat 
biscuit  or  Boas'  test-breakfast  should  be  given,  the 
stomach  having  been  thoroughly  washed  the  evening 
before. 

Uffelmann's  Test  for  Lactic  Acid. — ^Thoroughly  shake 
up  5  c.c.  of  filtered  stomach  fluid  with  50  c.c.  of  ether  for  at 
least  ten  minutes.  Collect  the  ether  and  evaporate  over  a 
water-bath.  Dissolve  the  residue  in  5  c.c.  of  water  and  test 
with  Uffelmann's  reagent  as  follows: 

In  a  test-tube  mix  3  drops  concentrated  solution  of  phenol 
and  3  drops  saturated  aqueous  solution  of  ferric  chlorid. 
Add  water  until  the  mixture  assumes  an  amethyst-blue 
color.  To  this  add  the  solution  to  be  tested.  The  appear- 
ance of  a  canary-yeUcnv  color  indicates  the  presence  of  lactic 
acid  (Plate  XI,  A,  A'). 


328 


THE   STOMACH 


Uffelmann's  test  may  be  applied  directly  to  the  stomach- 
contents  without  extracting  with  ether,  but  is  then  neither 
sensitive  nor  reliable,  because  of  the  phosphates,  sugars,  and 
other  interfering  substances  which  may  be  present. 

Kelling's  Test  {Simon^s  Modification) . — This  is  much  more 
satisfactory  than  Ufifelmann's.  To  a  test-tube  of  distilled 
water  add  sufficient  ferric  chlorid  solution  to  give  a  faint 
yellowish  tinge.  Pour  half  of  this  into  a 
second  test-tube  to  serve  as  a  control. 
To  the  other  add  a  small  amount  of  the 
gastric  juice.  Lactic  acid  gives  a  distinct 
yellow  color  which  is  readily  recognized 
by  comparison  with  the  control. 

Strauss'  Test  for  Lactic  Acid. — ^This 
is  a  good  test  for  clinical  work,  since  it 
gives  a  rough  idea  of  the  quantity  pres- 
ent and  is  not  sufficiently  sensitive  to 
respond  to  the  traces  of  lactic  acid  which 
some  test-meals  introduce.  Strauss'  in- 
strument (Fig.  io6)  is  essentially  a 
separating  funnel  with  a  mark  at  5  c.c. 
and  one  at  25  c.c.  Fill  to  the  5  c.c. 
mark  with  filtered  stomach  fluid,  and  to 
the  25  c.c.  mark  with  ether.  Shake 
thoroughly  for  ten  or  fifteen  minutes, 
let  stand  until  the  ether  separates,  and 
then,  by  opening  the  stop-cock,  allow  the 
gastric  juice  to  run  out.  Fill  to  the  25  c.c.  mark  with  water, 
and  add  2  drops  of  a  10  per  cent,  solution  of  ferric  chlorid. 
Shake  gently.  If  o.i  per  cent,  or  more  lactic  acid  be  present, 
the  water  will  assume  a  strong  greenish-yellow  color.  A 
slight  tinge  will  appear  with  0.05  per  cent. 

(4)  Pepsin  and  Pepsinogen. — Pepsinogen  itself  has 
no  digestive  power.    It  is  secreted  by  the  gastric  glands, 


Fip.  106. — Separatory 
funnel  for  Strauss'  lac- 
tic acid  test  (Sahli). 


EXAMINATION   OF   THE   GASTRIC   CONTENTS        329 

and  is  transformed  into  pepsin  by  the  action  of  a  free 
acid.  Although  pepsin  digests  proteins  best  in  the 
presence  of  free  hydrochloric  acid,  it  has  a  slight  diges- 
tive activity  in  the  presence  of  organic  or  combined 
hydrochloric  acids. 

The  amount  is  not  influenced  by  neuroses  or  circula- 
tory disturbances.  Absence  or  marked  diminution, 
therefore,  indicates  organic  disease  of  the  stomach.  This 
is  an  important  point  in  diagnosis  between  functional 
and  organic  conditions.  Pepsin  is  rarely  or  never  absent 
in  the  presence  of  free  hydrochloric  acid. 

Test  for  Pepsin  and  Pepsinogen. — ^With  a  cork-borer  cut 
small  cylinders  from  the  coagulated  white  of  an  egg,  and  cut 
these  into  disks  of  uniform  size.  The  egg  should  be  cooked 
very  slowly,  preferably  over  a  water-bath,  so  that  the  white 
may  be  readily  digestible.  The  disks  may  be  preserved  in 
glycerin,  but  must  be  washed  in  water  before  using. 

Place  a  disk  in  each  of  three  test-tubes. 

Into  tube  No.  i  put  10  c.c.  distilled  water,  5  grains  pepsin, 
U.  S.  P.,  and  3  drops  of  the  ofl&cial  dilute  hydrochloric  acid. 

Into  tube  No.  2  put  10  c.c.  filtered  gastric  juice. 

Into  tube  No.  3  put  10  c.c.  filtered  gastric  juice  and  3  drops 
dilute  hydrochloric  acid. 

Place  the  tubes  in  an  incubator  or  in  warm  water  for  three 
hours  or  longer.  At  intervals  observe  the  extent  to  which 
the  egg-albumen  has  been  digested.  This  is  recognized  by 
the  depth  to  which  the  disk  has  become  translucent. 

Tube  No.  I  is  used  for  comparison,  and  should  show  the 
effect  of  normal  gastric  juice. 

Digestion  of  the  egg  in  tube  No.  2  indicates  the  presence  of 
both  pepsin  and  free  hydrochloric  acid. 

When  digestion  fails  in  tube  No.  2  and  occurs  in  No.  3, 
pepsinogen  is  present,  having  been  transformed  into  pepsin 


33©  THE    STOMACH 

by  the  hydrochloric  acid  added.     Should  digestion  fail  in 
this  tube,  both  pepsin  and  pepsinogen  are  absent. 

(5)  Rennin  and  Renninogen. — Rennin  is  the  milk- 
curdling  ferment  of  the  gastric  juice.  It  is  derived  from 
renninogen  through  the  action  of  hydrochloric  acid. 
Lime  salts  also  possess  the  power  of  transforming  ren- 
ninogen into  the  active  ferment. 

Deficiency  of  rennin  has  the  same  significance  as 
deficiency  of  pepsin,  and  is  more  easily  recognized. 
Since  the  two  enzymes  are  almost  invariably  present  or 
absent  together,  the  test  for  rennin  serves  also  as  a  test 
for  pepsin. 

Test  for  Rennin. — Neutralize  5  c.c.  filtered  gastric  juice 
with  very  dilute  sodium  hydroxid  solution;  add  5  c.c.  fresh 
milk,  and  place  in  an  incubator  or  in  a  vessel  of  water  at 
about  40°  C.  Coagulation  of  the  milk  in  ten  to  fifteen 
minutes  shows  a  normal  amount  of  rennin.  Delayed  coagu- 
lation denotes  a  less  amount. 

Test  for  Renninogen. — To  5  c.c.  feebly  alkalinized  gastric 
juice  add  2  c.c.  of  i  per  cent,  calcium  chlorid  solution  and  5 
c.c.  fresh  milk,  and  place  in  an  incubator.  If  coagulation 
occurs,  renninogen  is  present. 

(6)  Peptid-splitting  Enzyme. — It  has  been  found  that 
in  cancer  of  the  stomach  there  may  be  present  a  patho- 
logic ferment  which  is  capable  of  splitting  peptids  into 
amino-acids.  No  such  ferment  is  present  normally, 
the  gastric  juice  being  incapable  of  carrying  digestion 
to  the  amino-acid  stage.  Neubauer  and  Fischer  have 
utilized  this  fact  for  the  diagnosis  of  gastric  cancer 
by  subjecting  the  dipeptid,  glycyl -tryptophan,  to  the 


EXAMINATION    OF   THE   GASTRIC   CONTENTS        33 1 

action  of  the  gastric  fluid  and  testing  for  the  presence 
of  the  amino-acid  tryptophan.  The  method  is  as 
follows: 

Place  10  c.c.  of  the  filtered  gastric  juice  and  about  i  c.c. 
of  glycyl-tryptophan  in  a  test-tube,  overlay  with  toluol  to 
prevent  bacterial  action,  and  place  in  an  incubator  at  about 
38°  C.  At  the  €nd  of  twenty-fovir  hours  pipet  off  a  few  cubic 
centimeters  and  test  for  tryptophan  as  follows:  Acidify  with 
a  few  drops  of  3  per  cent,  acetic  acid,  add  a  very  Uttle  bromin 
vapor  with  a  medicine-dropper,  and  shake.  The  appear- 
ance of  a  rose-red  color  shows  the  presence  of  tryptophan 
and  hence  of  the  peptid-splitting  ferment.  The  color  quickly 
disappears  if  too  much  bromin  is  added.  If  no  color  appears 
at  first,  add  more  bromin  vapor  in  small  quantities.  Only 
when  the  fluid  has  become  yellow  from  excess  of  bromin 
can  the  test  be  considered  negative. 

Before  applying  this  method,  the  stomach  fluid  must  be 
tested  for  pre-existing  tryptophan,  blood  (see  p.  333),  and 
bile  (see  p.  154).  Blood  and  pancreatic  juice  each  contain 
peptid-splitting  ferments,  and  pancreatic  juice  may  be  as- 
sumed to  be  present  if  bile  is  detected. 

Glycyl-trv-ptophan  can  be  purchased  in  bottles,  each 
containing  a  little  toluol  and  the  correct  amount  of  the  di- 
peptid  for  one  test.  The  gastric  juice  is  introduced  into  the 
bottle  to  the  level  of  a  mark  on  its  side  and  then  incubated. 
Such  an  outfit  is  called  a  "ferment  diagnosticum." 

Instead  of  glycyl-tryptophan,  Jacque  and  Woodyatt  and 
others  have  used  20  c.c.  sterilized  filtered  2  per  cent,  solu- 
tion of  Witte's  peptone  for  each  10  c.c.  of  stomach  fluid. 
They  then  estimate  amino-acids  in  10  c.c.  of  the  mixture 
before  incubating  and  in  10  c.c.  afterward,  using  a  formalin 
.method  which  is  practically  the  same  as  that  given  for 
ammonia  in  urine  (see  p.  127).  The  difference  between 
the  two  estimations  expresses  the  degree  of  peptolysis. 


332  THE   STOMACH 

The  value  of  the  test  is  impaired  by  Warfield's  dis- 
covery of  peptid-splitting  ferments  in  the  saliva.  Later 
workers  have  shown  that  much,  at  least,  of  the  peptolytic 
activity  of  the  saUva  is  due  to  ferments  of  leukocytes 
and  bacteria,  which  are  capable  of  splitting  proteins  as 
well  as  peptids.  The  chief  source  of  error,  however, 
appears  to  be  regurgitated  trypsin,  which  may  be  pres- 
ent in  the  absence  of  bile.  To  exclude  these  sources  of 
error  Friedman  and  Hamburger  propose  a  control  test 
for  proteolytic  ferments,  using  edestin  as  substrate.  If 
the  edestin  test  is  positive,  the  glycyl- tryptophan  test 
cannot  be  relied  upon.     It  is  performed  as  follows: 

Edestin  Test. — ^The  gastric  juice  is  filtered,  neutralized 

with   normal   Na2C03    solution,   using   phenolphthalein   as 

N 
indicator,  and  then  brought  to  an  alkalinity  equal  to  — 

Na^COa,  in  order  to  inactivate  pepsin.  Place  2  c.c.  of  a 
0.1  per  cent,  solution  of  edestin^  in  o.i  per  cent.  Na2C03  in 
each  of  four  test-tubes.  To  three  tubes  add  2  c.c,  i  c.c, 
and  0.5  c.c.  of  the  faintly  alkalinized  gastric  fluid,  reserving 
the  fourth  tube  as  a  control  and  adding  to  it  only  a  drop  of 
phenolphthalein  solution.  Place  the  four  tubes  in  an  incu- 
bator at  37°  C.  At  the  end  of  four  hours  exactly  neutralize 
the  contents  of  each  of  the  tubes  with  5  per  cent,  acetic  acid. 
When  the  neutral  point  is  reached  all  the  vmdigested  edestin 
will  be  precipitated.  The  degree  of  digestion  is  indicated  by 
the  amount  of  turbidity  compared  with  that  in  the  control 
tube.    Absence  of  turbidity  denotes  complete  digestion. 

(7)  Blood  is  present  in  the  vomitus  in  a  great  variety 
of  conditions.    When  found  in  the  fluid  removed  after 

*  Edestin  is  a  protein  extracted  from  hemp  seed.  It  can  be  pur- 
chased from  Eimer  and  Amend,  New  York. 


EXAMINATION   OF  THE    GASTRIC  CONTENTS        333 

a  test-meal,  it  commonly  points  toward  ulcer  or  car- 
cinoma. Blood  can  be  detected  in  nearly  one-half  of 
the  cases  of  gastric  cancer.  The  presence  of  swallowed 
blood  must  be  excluded. 

Test  for  Blood  in  Stomach-contents. — Extract  with  ether 
to  remove  fat.  To  10  c.c.  of  the  fat-free  fluid  add  a  few 
cubic  centimeters  of  glacial  acetic  acid  and  shake  the 
mixture  thoroughly  with  an  equal  volume  of  ether.  Separate 
the  ether  and  apply  to  it  the  guaiac  test  (see  p.  157);  or 
evaporate  and  apply  the  hemin  test  (see  p.  307)  to  the 
residue.  When  brown  particles  are  present  in  the  fluid,  the 
hemin  test  should  be  applied  directly  to  them. 

2.  Quantitative  Tests. — (i)  Total  Acidity. — The  acid- 
reacting  substances  which  contribute  to  the  total  acidity 
are  free  hydrochloric  acid,  combined  hydrochloric  acid, 
acid  salts,  mostly  phosphates,  and,  in  some  pathologic 
conditions,  the  organic  acids.  The  total  acidity  is  nor- 
mally about  50  to  75  degrees  (see  method  below),  or, 
when  estimated  as  hydrochloric  acid,  about  0.2  to  0.3 
per  cent.  With  Riegel's  or  Fischer's  test-meal  the  figures 
are  a  little  higher. 

Topfer's  Method  for  Total  Acidity. — ^In  an  evaporating 
dish  or  small  beaker  (an  "after-dinner"  cofi^ee-cup  is  a  very 
convenient  substitute)  take  10  c.c.  filtered  stomach-contents 
and  add  3  or  4  drops  of  the  indicator,  a  i  per  cent, 
alcoholic  solution  of  phenolphthalein.  When  the  quantity  of 
stomach  fluid  is  small,  5  c.c.  may  be  used,  but  results  are  less 
^accurate  than  with  a  larger  amount.  Add  decinormal  solu- 
tion of  sodium  hydroxid  drop  by  drop  from  a  buret,  until 
the  fluid  assumes  a  rose-red  color  which  does  not  become 


334  THE   STOMACH 

deeper  upon  addition  of  another  drop  (Plate  XII,  A,  A'). 
In  ordinary  titrations  the  end-point  is  the  appearance  of  the 
first  permanent  pink,  but  owing  to  interaction  of  phosphates 
it  is  advised  (Wood)  to  carry  the  titration  of  gastric  juice 
a  little  farther,  as  here  indicated.  When  this  point  is  reached, 
all  the  acid  has  been  neutralized.  The  end  reaction  will  be 
sharper  if  the  fluid  be  saturated  with  sodium  chlorid.  A 
sheet  of  white  paper  beneath  the  beaker  facilitates  recog- 
nition of  the  color  change. 

In  clinical  work  the  amount  of  acidity,  is  expressed  by  the 
number  of  cubic  centimeters  of  the  decinormal  sodium  hy- 
droxid  solution  which  would  be  required  to  neutralize  lOO  c.c. 
of  the  gastric  juice,  each  cubic  centimeter  representing  one 
degree  of  acidity.  Hence,  multiply  the  number  of  cubic  centi- 
meters of  decinormal  solution  required  to  neutralize  the  lo 
c.c.  of  stomach  fluid  by  lo.  This  gives  the  number  of  de- 
grees of  acidity.  The  amount  may  be  expressed  in  terms  of 
hydrochloric  acid,  if  one  remember  that  each  degree  is  equiva- 
lent to  0.00365  per  cent,  hydrochloric  acid.  Some  one  sug- 
gests that  this  is  the  mmiber  of  days  in  the  year,  the  last 
figure,  5,  indicating  the  nimiber  of  decimal  places. 

Example. — Suppose  that  7  c.c.  of  decinormal  solution  were 
required  to  bring  about  the  end  reaction  in  10  c.c.  gastric 
juice;  then  7  X  10  =  70  degrees  of  acidity;  and,  expressed  in 
terms  of  hydrochloric  acid,  70  X  0.00365  =  0.255  P^  f^^^- 

Preparation  of  decinormal  solutions  is  described  in  text- 
books on  chemistry.  The  practitioner  will  find  it  best  to 
have  them  made  by  a  chemist,  or  to  purchase  from  a  chemic 
supply  house.  Preparation  of  an  approximately  decinormal 
solution  is  described  on  page  549. 

(2)  Hydrochloric  Acid. — After  the  Ewald  and  Boas 
test-breakfasts  the  amount  of  free  hydrochloric  acid 
varies  normally  between  25  and  50  degrees,  or  about  o.i 


PLATE  XII 


N_ 


U 


<~ 


A,  Gastric  fluid  to  which  a  i  per  cent,  solution  of  phenolphthalein 
has  been  added;  B,  gastric  fluid  to  which  a  i  per  cent,  solution  of  alizarin 
has  been  added;  C,  gastric  fluid  to  which  a  0.5  per  cent,  solution  of 
dimethylamido-azobenzol  has  been  added;  A',  A  after  titration  with  a 
decinormal  solution  of  sodium  hydroxid;  B',  B  after  titration  with  a 
decinormal  solution  of  sodium  hydroxid;  C,  C  after  titration  with  a 
decinormal  solution  of  sodium  hydroxid  (Boston). 


EXAMINATION   OF   THE   GASTRIC   CONTENTS        335 

to  0.2  per  cent.  In  disease  it  may  go  considerably 
higher  or  may  be  absent  altogether. 

When  the  amount  of  free  hydrochloric  acid  is  normal, 
organic  disease  of  the  stomach  probably  does  not  exist. 

Increase  of  free  hydrochloric  acid  above  50  degrees 
(hyperchlorhydria)  generally  indicates  a  neurosis,  but 
also  occurs  in  most  cases  of  gastric  ulcer  and  beginning 
chronic  gastritis. 

Decrease  of  free  hydrochloric  acid  below  25  degrees 
(hypochlorhydria)  occurs  in  some  neuroses,  chronic  gas- 
tritis, early  carcinoma,  and  most  conditions  associated 
with  general  systemic  depression.  Marked  variation  in 
the  amount  at  successive  examinations  strongly  suggests 
a  neurosis.  Too  low  values  are  often  obtained  at  the 
first  examination,  the  patient's  dread  of  the  introduction 
of  the  tube  probably  inhibiting  secretion. 

A  hsence  of  free  hydrochloric  acid  (achlorhydria)  occurs 
in  most  cases  of  gastric  cancer  and  far-advanced  chronic 
gastritis,  in  many  cases  of  pernicious  anemia,  and  some- 
times in  hysteria  and  pulmonary  tuberculosis. 

The  presence  of  free  hydrochloric  acid  presupposes  a 
normal  amount  of  combined  hydrochloric  acid,  hence  the 
combined  need  not  be  estimated  when  the  free  acid  has 
been  found.  When,  however,  free  hydrochloric  acid  is 
absent,  it  is  important  to  know  whether  any  acid  is 
secreted,  and  an  estimation  of  the  combined  acid  then 
becomes  of  great  value.  The  normal  average  after  an 
Ewald  breakfast  is  about  10  to  15  degrees,  the  quantity 
depending  upon  the  amount  of  protein  in  the  test-meal. 
Somewhat  higher  figures  are  obtained  after  a  Riegel  or 
Fischer  test-meal. 


336  THE   STOMACH 

Topfer's  Method  for  Free  Hydrochloric  Acid. — In  a  beaker 
take  lo  c.c.  filtered  stomach  fluid  and  add  4  drops  of  the 
indicator,  a  0.5  per  cent,  alcoholic  solution  of  dimethyl- 
amido-azobenzol.  A  red  color  instantly  appears  if  free  hydro- 
chloric acid  be  present.  Add  decinormal  sodium  hydroxid 
solution,  drop  by  drop  from  a  buret,  until  the  last  trace  of  red 
just  disappears,  and  a  canary-yellow  color  takes  its  place 
(Plate  XII,  C,  C).  Read  off  the  number  of  cubic  centimeters 
of  decinormal  solution  added,  and  calculate  the  degrees,  or 
percentage  of  free  hydrochloric  acid,  as  in  Topfer's  method 
for  total  acidity. 

When  it  is  impossible  to  obtain  sufficient  fluid  for  all  the 
tests,  it  will  be  found  convenient  to  estimate  the  free  hydro- 
chloric acid  and  total  acidity  in  the  same  portion.  After 
finding  the  free  hydrochloric  acid  as  just  described,  add  4 
drops  phenolphthalein  solution,  and  continue  the  titration. 
The  amount  of  decinormal  solution  used  in  both  titrations 
indicates  the  total  acidity. 

Topfer's  Method  for  Combined  Hydrochloric  Acid. — In 
a  beaker  take  10  c.c.  filtered  gastric  juice  and  add  4  drops 
of  the  indicator,  a  i  per  cent,  aqueous  solution  of  sodium 
alizarin  sulphonate.  Titrate  with  decinormal  sodium  hy- 
droxid until  the  appearance  of  a  bluish-violet  color  which 
does  not  become  deeper  upon  addition  of  another  drop  (Plate 
XII,  B,  B').  It  is  difficult,  without  practice,  to  determine 
when  the  right  color  has  been  reached.  A  reddish  violet 
appears  first.  The  shade  which  denotes  the  end  reaction  can 
be  produced  by  adding  2  or  3  drops  of  the  indicator  to  5  c.c. 
of  I  per  cent,  sodium  carbonate  solution. 

Calculate  the  number  of  cubic  centimeters  of  decinormal 
solution  which  would  be  required  for  100  c.c.  of  stomach  fluid. 
This  gives,  in  degrees,  all  the  acidity  except  the  combined  hydro- 
chloric acid.  The  combined  hydrochloric  acid  is  then  found 
by  deducting  this  amount  from  the  total  acidity,  which  has 
been  previously  determined. 


EXAMINATION   OF   THE   GASTRIC  CONTENTS        337 

Example. — Suppose  that  5  c.c.  of  decinoraial  solution  were 
required  to  produce  the  purple  color  in  10  c.c.  gastric  juice; 
then  5  X  10  =  50  =  a//  the  acidity  except  combined  hydrochloric 
acid.  Suppose,  now,  that  the  total  acidity  has  already  been 
found  to  be  70  degrees;  then  70—50=  20  degrees  of  combined 
hydrochloric  acid;  and  20X0.00365  =  0.073  per  cent. 

When  free  hydrochloric  acid  is  absent,  it  is  probably 
more  helpful  to  estimate  the  acid  deficit  than  the  com- 
bined hydrochloric  acid.  The  acid  deficit  shows  how 
far  the  acid  secreted  by  the  stomach  falls  short  of  satu- 
rating the  protein  (and  bases)  of  the  meal.  It  repre- 
sents the  amount  of  hydrochloric  acid  which  must  be 
added  to  the  fiuid  before  a  test  for  free  hydrochloric 
acid  can  be  obtained.     It  is  determined  by  titrating 

with  —    hydrochloric  acid,  using  dimethyl-amido-azo- 

benzol  as  indicator,  until  the  fluid  assumes  a  red  color. 
The  amount  of  deficit  is  expressed  by  the  number  of 
cubic  centimeters  of  the  decinormal  solution  required 
for  100  c.c  of  the  stomach  fluid. 

(3)  Organic  Acids. — There  is  no  simple  direct  quan- 
titative method.  After  the  total  acidity  has  been  deter- 
mined, organic  acids  may  be  removed  from  another 
portion  of  the  gastric  filtrate  by  shaking  thoroughly 
with  an  equal  volume  of  neutral  ether,  allowing  the 
fluids  to  separate,  and  repeating  this  process  until  the 
gastric  fluid  has  been  extracted  with  eight  or  ten  times 
its  volume  of  ether.  The  total  acidity  is  then  deter- 
mined, and  the  difference  between  the  two  determina- 
tions indicates  the  amount  of  organic  acids. 

(4)  Pepsin. — No  direct  method  is  available.  The 
following  are  sufficient  for  clinical  purposes: 

22 


338  THE   STOMACH 

(i)  Hammerschlag's  Method. — To  the  white  of  an  egg  add 
twelve  times  its  volume  of  0.4  per  cent,  hydrochloric  acid 
(dilute  hydrochloric  acid,  U.  S.  P.,  4  c.c;  water,  96  c.c), 
mix  well,  and  filter.  This  gives  a  i  per  cent,  egg-albumen 
solution.  Take  10  c.c.  of  this  solution  in  each  of  three  tubes 
or  beakers.  To  A  add  5  c.c.  gastric  juice;  to  B,  5  c.c.  water 
with  0.5  gm.  pepsin;  to  C,  5  c.c.  water  only.  Place  in  an 
incubator  for  an  hour  and  then  determine  the  amount  of 
albumin  in  each  mixture  by  Esbach's  method.  Tube  C  shows 
the  amount  of  albumin  in  the  test-solution.  The  difference 
between  C  and  B  indicates  the  amount  of  albumin  which 
would  be  digested  by  normal  gastric  juice.  The  difference 
between  C  and  A  gives  the  albumin  which  is  digested  by  the 
fluid  under  examination.  Schiitz  has  shown  that  the  amounts 
of  pepsin  in  two  fluids  are  proportionate  to  the  squares  of  the 
products  of  digestion.  Thus,  if  the  amounts  of  albumin  di- 
gested in  tubes  A  and  B  are  to  each  other  as  2  is  to  4,  the 
amovmts  of  pepsin  are  to  each  other  as  4  is  to  16. 

Certain  sources  of  error  can  be  eliminated  by  diluting  the 
gastric  juice  several  times  before  testing.  The  most  impor- 
tant of  these  are  that  the  law  of  Schiitz  holds  good  only  for 
comparatively  dilute  solutions,  and  that  the  products  of 
peptic  activity  inhibit  digestion. 

(2)  Mett's  method  is  generally  preferred  to  the  preceding. 
Put  three  or  four  Mett's  tubes  about  2  cm.  long  into  a  small 
beaker  with  diluted  gastric  juice  (i  c.c.  of  the  filtrate  plus  15 
c.c.  twentieth-normal  hydrochloric  acid).  Place  in  an  incu- 
bator for  twenty-four  hours,  and  then  measure  as  accurately 
as  possible  in  millimeters  the  column  which  has  been  digested, 
using  a  millimeter  scale  and  a  hand  lens  or,  better,  a  low 
power  of  the  microscope  and  an  eye-piece  micrometer. 
Square  the  average  length  of  this  column  (law  of  Schiitz) 
and  multiply  by  the  degree  of  dilution,  16.  The  maximum 
figure  obtained  in  this  way  is  256,  representing  a  digested 
colunm  of  4  mm. 


EXAMESTATION   OF   THE   GASTRIC   CONTENTS        339 

Prepare  Mett's  tubes  as  follows: 

Beat  up  slightly  the  whites  of  one  or  two  eggs  and  filter. 
Pour  into  a  wide  test-tube  and  stand  in  this  a  number  of 
capillary  glass  tubes,  i  to  2  mm.  in  diameter.  When  the 
tubes  are  filled,  plug  their  ends  with  bread  crumbs,  and  coag- 
ulate the  albumin  by  heating  in  water  just  short  of  boiling. 
Dip  the  ends  of  the  tubes  in  melted  paraffin  and  preserve  imtil 
needed.  Bubbles,  if  present,  will  probably  disappear  in  a 
few  days.  When  wanted  for  use,  cut  the  tubes  into  lengths 
of  about  '2  cm.  Discard  any  in  which  the  albumin  has  sep- 
arated from  the  wall. 


D.   Microscopic  Examination 

A  drop  of  unfiltered  stomach-contents  is  placed  upon 
a  slide,  covered  with  a  cover-glass,  and  examined  with 
the  i6-mm.  and  4- mm.  objectives.  A  drop  of  Lugol's 
solution  allowed  to  run  under  the  cover  will  aid  in  dis- 
tinguishing the  various  structures.  As  a  rule,  the  micro- 
scopic examination  is  of  little  value. 

Under  normal  conditions  little  is  to  be  seen  except 
great  numbers  of  starch-granules,  with  an  occasional 
epithelial  cell,  yeast-cell,  or  bacterium.  Starch-granules 
are  recognized  by  their  concentric  striations  and  the 
fact  that  they  stain  blue  with  iodin  solutions  when  undi- 
gested, and  reddish,  due  to  erythrodextrin,  when  partially 
digested. 

Pathologically,  remnants  of  food  from  previous  meals, 
red  blood-corpuscles,  pus-cells,  sarcinse,  and  excessive 
numbers  of  yeast-cells  and  bacteria  may  be  encountered 
{Fig.  107). 

Remnants  of  food  from  previous  meals  indicate 
deficient  gastric  motility. 


34©  THE   STOMACH 

Red  Blood-corpuscles. — Blood  is  best  recognized  by 
the  chemic  tests  already  given.  The  corpuscles  some- 
times retain  a  fairly  normal  appearance,  but  are  generally 
so  degenerated  that  only  granular  pigment  is  left.  When 
only  a  few  fresh-looking  corpuscles  are  present,  they 
usually  come  from  irritation  of  the  mucous  membrane 
by  the  tube. 


Fig.  107. — General  view  of  the  gastnc  contents:  a,  Squamous  epithelial  cells  from 
esophagus  and  mouth;  6,  leukocytes;  c,  cylindric  epithelial  cells;  d,  muscle-fibers;  e,  fat- 
droplets  and  fat-crystals;  /,  starch-granules;  g,  chlorophyl-containing  vegetable  mat- 
ters; h,  vegetable  spirals;  i,  bacteria;  k,  sarcinae;  /,  budding  (yeast)  fungi  Qakob). 


Pus-cells. — Pus  is  rarely  encountered  in  the  fluid 
removed  after  a  test-meal.  Considerable  numbers  of 
pus-corpuscles  have  been  found  in  some  cases  of  gastric 
cancer.  The  corpuscles  are  usually  partially  digested, 
so  that  only  the  nuclei  are  seen.  Swallowed  sputum 
must  always  be  considered. 

Sarcinae. — These  are  small  spheres  arranged  in  cuboid 
groups,  often  compared  to  bales  of  cotton.  They  fre- 
quently form  large  clumps  and  are  easily  recognized. 
They  stain  brown  with  iodin  solution.    They  signify  fer- 


EXAMINATION   OF   THE   GASTRIC   CONTENTS        34 1 

mentation.  Their  presence  is  strong  evidence  against 
the  existence  of  gastric  cancer,  in  which  disease  they 
rarely  occur. 

Yeast-cells. — As  already  stated,  a  few  yeast-cells 
may  be  found  under  normal  conditions.  The  presence 
of  considerable  numbers  is  evidence  of  fermentation. 
Their  appearance  has  been  described  (see  p.  208).  They 
stain  brown  with  iodin  solution. 

Bacteria. — Numerous  bacteria  may  be  encountered, 
especially  in  the  absence  of  free  hydrochloric  acid.    The 


Fig.  108. — Boas-Oppler  bacillus  from  case  of  gastric  cancer  (Boston). 

Boas-Oppler  bacillus  is  the  only  one  of  special  signifi- 
cance. It  occurs  in  the  majority  of  cases  of  cancer,  and 
is  rarely  found  in  other  conditions.  Carcinoma  probably 
furnishes  a  favorable  medium  for  its  growth. 

These  bacilli  (Fig.  108)  are  large  (5  to  10  ^  long), 
non-motile,  and  usually  arranged  in  clumps  or  end  to 
end  in  zig-zag  chains.  They  stain  brown  with  iodin 
solution,  which  distinguishes  them  from  Leptothrix  buc- 
calis  (see  p.  433),  which  is  not  infrequently  swallowed, 
and  hence  found  in  stomach  fluid.    They  also  stain  by 


342  THE   STOMACH 

Gram's  method.  They  are  easily  seen  with  the  4-mm. 
objective  in  unstained  preparations,  but  are  best  recog- 
nized with  the  oil  lens,  after  drying  some  of  the  fluid 
upon  a  cover-glass,  fixing,  and  staining  with  a  simple 
bacterial  stain  or  by  Gram's  method. 

A  few  large  non-motile  bacilli  are  frequently  seen;  they 
cannot  be  called  Boas-Oppler  bacillus  unless  they  are 
numerous  and  show  something  of  the  typical  arrange- 
ment. 

E.   The  Gastric  Contents  in  Disease 

In  the  diagnosis  of  stomach  disorders  the  practitioner 
must  be  cautioned  against  relying  too  much  upon  exam- 
inations of  the  stomach-contents.  A  fifst  examination 
is  especially  unreKable.  Even  when  repeated  examina- 
tions are  made,  the  laboratory  findings  must  never  be 
considered  apart  from  the  clinical  signs. 

The  more  characteristic  findings  in  certain  disorders 
are  suggested  here: 

1 .  Dilatation  of  the  Stomach. — Evidences  of  retention 
and  fermentation  are  the  chief  characteristics  of  this 
condition.  Hydrochloric  acid  is  commonly  diminished. 
Pepsin  may  be  normal  or  slightly  diminished.  Lactic 
acid  may  be  detected  in  small  amounts,  but  is  usually 
absent  when  the  stomach  has  been  washed  before  giving 
the  test-meal.  Both  motility  and  absorptive  power  are 
deficient.  The  microscope  commonly  shows  sarcinae, 
bacteria,  and  great  numbers  of  yeast-cells.  Remnants 
of  food  from  previous  meals  can  be  detected  with  the 
naked  eye  or  microscopically. 

2.  Gastric  Neuroses.— The  findings  are  variable. 
Successive  examinations  may  show  normal,  increased, 


EXAMINATION    OF   THE   GASTRIC   CONTENTS        343 

or  diminished  hydrochloric  acid,  or  even  entire  absence 
of  the  free  acid.     Pepsin  is  usually  normal. 

In  the  neurosis  characterized  by  continuous  hyperse- 
cretion (gastrosuccorrhea) ,  40  c.c.  or  more  of  gastric 
juice  can  be  obtained  from  the  fasting  stomach.  Should 
the  fluid  contain  food-particles,  it  is  probably  the  result 
of  retention,  not  hypersecretion. 

3.  Chronic  Gastritis. — Free  hydrochloric  acid  may  be 
increased  in  early  cases.  It  is  generally  diminished  in 
well-marked  cases,  and  is  often  absent  in  advanced 
cases.  Lactic  acid  is  often  present  in  traces,  rarely  in 
notable  amount.  Secretion  of  pepsin  and  rennin  is 
always  diminished  in  marked  cases.  Mucus  is  fre- 
quently present,  and  is  very  significant  of  the  disease. 
Motility  and  absorption  are  generally  deficient.  Small 
fragments  of  mucous  membrane  may  be  found,  and  when 
examined  by  a  pathologist,  may  occasionally  establish 
the  diagnosis. 

4.  Achylia  Gastrica  (Atrophic  Gastritis). — This  con- 
dition may  be  a  terminal  stage  of  chronic  gastritis.  It  is 
sometimes  associated  with  the  blood-picture  of  perni- 
cious anemia.  It  gives  a  great  decrease,  and  sometimes 
entire  absence  of  hydrochloric  acid  and  ferments.  The 
total  acidity  may  be  as  low  as  i  or  2  degrees.  Small 
amounts  of  lactic  acid  may  be  present.  Absorption  and 
motility  are  usually  not  affected  greatly. 

5.  Gastric  Carcinoma. — As  far  as  the  laboratory 
examination  goes,  the  cardinal  signs  of  this  disease  are 
absence  of  free  hydrochloric  acid  and  presence  of  a 
pep tid- splitting  ferment,  of  lactic  acid,  and  of  the  Boas- 
Oppler  bacillus.  These  findings  are,  however,  by  no 
means  constant. 


344  THE    STOMACH 

It  is  probable  that  some  substance  is  produced  by  the 
cancer  which  neutralizes  the  free  hydrochloric  acid,  and 
thus  causes  it  to  disappear  earlier  than  in  other  organic 
diseases  of  the  stomach.  The  peptid-splitting  ferment 
(see  p.  330)  is  also  probably  a  product  of  the  cancer. 

The  presence  of  lactic  acid  is  possibly  the  most  sug- 
gestive single  symptom  of  gastric  cancer.  In  the  great 
majority  of  cases  its  presence  in  notable  amoxmt  (o.i 
per  cent,  by  Strauss'  method)  after  Boas'  breakfast,  the 
stomach  having  been  washed  the  evening  before,  war- 
rants a  tentative  diagnosis  of  malignancy. 

Carcinoma  seems  to  furnish  an  especially  favorable 
medium  for  the  growth  of  the  Boas-Oppler  bacillus, 
hence  this  micro-organism  is  frequently  present. 

Blood  can  be  detected  in  the  stomach  fluid  by  the 
chemic  tests  in  nearly  one-half  of  the  cases,  and  is  more 
common  when  the  new  growth  is  situated  at  the  pylorus. 
Blood  is  present  in  the  stool  in  nearly  every  case. 

E\ddences  of  retention  and  fermentation  are  the  rule 
in  pyloric  cancer.  Tumor  particles  are  sometimes  found 
late  in  the  disease. 

6.  Gastric  Ulcer. — There  is  excess  of  free  hydrochloric 
acid  in  about  one-half  of  the  cases.  In  other  cases  the 
acid  is  normal  or  diminished.  Blood  is  often  present. 
The  diagnosis  must  be  based  largely  upon  the  clinical 
symptoms,  and  where  ulcer  is  strongly  suspected,  it  is 
probably  unwise  to  use  the  stomach-tube. 

n.  ADDITIONAL   EXAMINATIONS    WHICH  GIVE  INFOR- 
MATION AS  TO  THE  CONDITION  OF  THE  STOMACH 

1 .  Absorptive  Power  of  the  Stomach. — This  is  a  very 
unimportant  function,  only  a  few  substances  being  ab- 


EXAMINATIONS  AS  TO  THE  CONDITION  OF  STOMACH    345 

sorbed  in  the  stomach.  It  is  delayed  in  most  organic 
diseases  of  the  stomach,  especially  in  dilatation  and 
carcinoma,  but  not  in  neuroses.  The  test  has  little 
practical  value. 

Give  the  patient,  upon  an  empty  stomach,  a  3 -grain  cap- 
sule of  potassium  iodid  with  a  glass  of  water,  taking  care 
that  none  of  the  drug  adheres  to  the  outside  of  the  capsule. 
At  intervals  test  the  saliva  for  iodids  by  moistening  starch- 
paper  with  it  and  touching  with  yellow  nitric  acid.  A  blue 
color  shows  the  presence  of  an  iodid,  and  appears  normally 
in  ten  to  fifteen  minutes  after  ingestion  of  the  capsule.  A 
longer  time  denotes  delayed  absorption. 

Starch-paper  is  prepared  by  soaking  filter-paper  in  boiled 
starch  and  drying. 

2.  Motor  Power  of  the  Stomach. — This  refers  to  the 
rapidity  with  which  the  stomach  passes  its  contents  on 
into  the  intestines.  It  is  very  important :  intestinal  diges- 
tion can  compensate  for  insufficient  or  absent  stomach 
digestion  only  so  long  as  the  motor  power  is  good. 

MotiHty  is  impaired  to  some  extent  in  chronic  gas- 
tritis. It  is  especially  deficient  in  dilatation  of  the  stom- 
ach due  to  atony  of  the  gastric  wall  or  to  pyloric  obstruc- 
tion, either  benign  or  malignant.  It  is  increased  in  most 
conditions  with  hyperchlorhydria. 

The  best  evidence  of  deficient  motor  power  is  the 
detection  of  food  in  the  stomach  at  a  time  when  it  should 
be  empty,  e.  g.,  before  breakfast  in  the  morning.  A 
special  test-meal  containing  easily  recognized  materials 
{e.  g.,  rice  pudding  with  currants)  is  sometimes  given  and 
removed  at  the  end  of  six  or  seven  hours.  When  more 
than  60  c.c.  of  fluid  are  obtained  with  the  tube  one  hour 


346  THE   STOMACH 

after  an  Ewald  breakfast,  deficient  motility  may  be 
inferred. 

Ewald's  salol  test  is  scarcely  so  reliable  as  the  above.  It 
depends  upon  the  fact  that  salol  is  not  absorbed  until  it 
reaches  the  intestines  and  is  decomposed  by  the  alkaline 
intestinal  juices. 

The  patient  is  given  15  grains  of  salol  with  a  test-breakfast, 
and  the  urine,  passed  at  intervals  thereafter,  is  tested  for 
salicyluric  acid.  A  few  drops  of  10  per  cent,  ferric  chlorid 
solution  are  added  to  a  small  quantity  of  the  urine.  A  violet 
color  denotes  the  presence  of  salicyluric  acid.  It  appears 
normally  in  sixty  to  seventy-five  minutes  after  ingestion  of  the 
salol.    A  longer  time  indicates  impaired  motor  power. 

3.  To  Determine  Size  and  Position  of  Stomach. — 

After  removing  the  test-meal,  while  the  tube  is  still  in 
place  force  quick  puffs  of  air  into  the  stomach  by  com- 
pression of  the  bulb.  The  puffs  can  be  clearly  heard 
with  a  stethoscope  over  the  region  of  the  stomach,  and 
nowhere  else. 

If  desired,  the  patient  may  be  given  a  dram  of  sodium 
bicarbonate  in  solution,  followed  immediately  by  the 
same  amount  of  tartaric  acid,  also  in  solution;  or  he  may 
take  the  two  parts  of  a  SeidUtz  powder  separately.  The 
carbon  dioxid  evolved  distends  the  stomach,  and  its 
outline  can  easily  be  determined  by  percussion. 

4.  Sahli's  Desmoid  Test  of  Gastric  Digestion. — Two 
pills,  one  containing  o.i  gram  iodoform,  the  other  0.05 
gram  methylene-blue,  are  wrapped  in  little  bags  made  of 
thin  sheets  of  rubber  and  tied  with  a  string  of  raw  cat- 
gut, No.  00.  The  bags  must  be  carefully  folded  and  tied. 
Before  use  they  should  be  placed  for  a  time  in  water. 


EXAMINATIONS  AS  TO  THE  CONDITION   OF  STOMACH    347 

If  they  float  or  if  any  of  the  methylene-blue  escapes  and 
colors  the  water  they  are  useless  for  the  test. 

The  patient  swallows  the  two  bags  with  the  aid  of  a 
little  water  during  the  noon  meal,  and  the  urine  is  tested 
at  intervals  thereafter.  According  to  Sahli,  the  catgut 
is  digested  by  gastric  juice  and  not  by  pancreatic  or 
intestinal  juices.  If  gastric  digestion  is  normal,  iodin 
and  methylene-blue  can  be  detected  in  the  urine  in  the 
afternoon  or  evening  of  the  same  day.  The  reaction 
may  occur  when  digestion  is  very  poor,  provided  gastric 
motility  is  diminished,  but  it  is  then  delayed.  If  the 
reaction  does  not  appear,  gastric  digestion  has  not 
occurred. 

Methylene-blue  is  recognized  in  the  urine  by  the  green  or 
blue  color  which  it  imparts.  It  is  sometimes  eliminated  as 
a  chromogen,  in  which  case  a  little  of  the  urine  must  be 
acidified  with  acetic  acid  and  boiled  to  bring  out  the  color. 

To  detect  the  iodin,  some  of  the  urine  is  decolorized  by 
gently  heating  and  filtering  through  animal  charcoal.  To 
ID  c.c.  are  then  added  i  c.c.  dilute  sulphuric  acid,  and  0.5 
c.c.  of  a  I  per  cent,  solution  of  sodium  nitrite  and  2  c.c.  of 
chloroform.  Upon  shaking,  a  rose  color  will  be  imparted  to 
the  chloroform  if  iodin  be  present.  Another  method  of 
testing  for  iodin  is  given  on  page  168. 


CHAPTER  V 

THE  FECES 

As  commonly  practised,  an  examination  of  the  feces  is 
limited  to  a  search  for  intestinal  parasites  or  their  ova. 
Much  of  value  can,  however,  be  learned  from  other  simple 
examinations,  particularly  a  careful  inspection.  Any- 
thing approaching  a  complete  analysis  is,  on  the  other 
hand,  a  waste  of  time  for  the  clinician. 

The  normal  stool  is  a  mixture  of — (a)  water;  (6) 
undigested  and  indigestible  remnants  of  food,  as  starch- 
granules,  particles  of  meat,  vegetable  cells  and  fibers, 
etc.;  (c)  digested  foods,  carried  out  before  absorption 
can  take  place;  (d)  products  of  the  digestive  tract,  as 
altered  bile-pigments,  mucus,  etc.;  (e)  products  of  de- 
composition, as  indol,  skatol,  fatty  acids,  and  various 
gases;  (/)  epithelial  cells  shed  from  the  wall  of  the  in- 
testinal canal;  (g)  harmless  bacteria,  which  are  always 
present  in  enormous  numbers. 

Pathologically,  we  may  find  abnormal  amounts  of 
normal  constituents,  blood,  pathogenic  bacteria,  animal 
parasites  and  their  ova,  and  biliary  and  intestinal  con- 
cretions. 

The  stool  to  be  examined  should  be  passed  into  a  clean 
vessel,  without  admixture  of  urine.  The  examination 
should  not  be  delayed  more  than  a  few  hours,  owing  to 
the  changes  caused  by  decomposition.  The  offensive 
odor  can  be  partially  overcome  with  turpentine  or  5  per 
cent,  phenol.   When  search  for  amebae  is  to  be  made,  the 

348 


MACROSCOPIC   EXAMINATION  349 

vessel  must  be  warm,  and  the  stool  kept  warm  until 
examined;  naturally,  no  disinfectant  can  be  used.  For 
other  protozoa  a  saline  cathartic  may  be  given  and  the 
second  stool  examined.  The  first  -stool  is  usually  too 
solid,  and  the  later  ones  too  greatly  diluted. 

L  MACROSCOPIC  EXAMINATION 

1.  Quantity. — The  amount  varies  greatly  with  diet 
and  other  factors.  The  average  is  about  loo  to  150  gm. 
in  twenty-four  hours. 

2.  Frequency. — One  or  two  stools  in  twenty-four  hours 
may  be  considered  normal,  yet  one  in  three  or  four  days 
is  not  uncommon  with  healthy  persons.  The  individual 
habit  should  be  considered  in  every  case. 

3.  Form  and  Consistence. — Soft,  mushy,  or  Uquid 
stools  follow  cathartics  and  accompany  diarrhea.  Co- 
pious, purely  serous  discharges  without  fecal  matter 
are  significant  of  Asiatic  cholera,  although  sometimes 
observed  in  other  conditions.  Hard  stools  accompany 
constipation.  Rounded  scybalous  masses  are  common 
in  habitual  constipation,  and  indicate  atony  of  the  mus- 
cular coat  of  the  colon.  Flattened,  ribbon-hke  stools  re- 
sult from  some  obstruction  in  the  rectum,  generally  a 
tumor  or  a  stricture  from  a  healed  ulcer,  most  commonly 
syphilitic.  When  bleeding  piles  are  absent,  blood- 
streaks  upon  such  a  stool  point  to  carcinoma. 

4.  Color. — The  normal  light  or  dark-brown  color  is  due 
chiefly  to  hydrobilirubin,  which  is  formed  from  bihrubin 
by  reduction  processes  in  the  intestine,  largely  the  result 
of  bacterial  activity.  The  stools  of  infants  are  yellow, 
owing  partly  to  their  milk  diet  and  partly  to  the  presence 
of  unchanged  bilirubin. 


35°  THE   FECES 

Diet  and  drugs  cause  marked  changes:  milk,  a  light 
yellow  color;  cocoa  and  chocolate,  dark  gray;  various 
fruits,  reddish  or  black;  iron  and  bismuth,  dark  brown  or 
black;  hematoxylin,  red,  etc. 

Pathologically,  the  color  is  important.  A  golden  yel- 
low is  generally  due  to  unchanged  bilirubin.  Green 
stools  are  not  uncommon,  especially  in  diarrheas  of  child- 
hood. They  are  sometimes  met  in  apparently  healthy 
infants,  alternating  with  normal  yellow  stools,  and  have 
little  significance  unless  accompanied  by  symptoms. 
The  color  is  due  to  biliverdin  or,  sometimes,  to  chromo- 
genic  bacteria.  Putty-colored  or  "acholic"  stools  occur 
when  bile  is  deficient,  either  from  obstruction  to  outflow 
or  from  deficient  secretion.  The  color  is  due  less  to  ab- 
sence of  bile-pigments  than  to  presence  of  fat.  Similar 
stools,  which  manifestly  consist  largely  of  fat,  are  com- 
mon in  conditions  like  tuberculous  peritonitis,  which 
interfere  with  absorption  of  fats,  and  in  pancreatic  dis- 
ease. 

Notable  amounts  of  blood  produce  tarry  black  stools 
when  the  source  of  the  hemorrhage  is  the  stomach  or 
upper  intestine,  and  a  dark  brown  or  bright  red  as  the 
source  is  nearer  the  rectum.  When  diarrhea  exists  the 
color  may  be  red,  even  if  the  source  of  the  blood  is  high 
up.  Red  streaks  of  blood  upon  the  outside  of  the  stool 
are  due  to  lesions  of  rectum  or  anus. 

5.  Odor. — Products  of  decomposition,  chiefly  indol 
and  skatol,  are  responsible  for  the  normal  offensive  odor. 
A  sour  odor  is  normal  for  nursing  infants,  and  is  noted  in 
mild  diarrheas  of  older  children.  In  the  severe  diarrheas 
of  childhood  a  putrid  odor  is  common.  In  adults,  stools 
emitting  a  very  foul  stench  are  suggestive  of  malignant 


MACROSCOPIC   EXAMINATION  35 1 

or  syphilitic  ulceration  of   the   rectum  or  gangrenous 
dysentery. 

6.  Mucus.— Excessive  quantities  of  mucus  are  easily 
detected  with  the  naked  eye,  and  signify  irritation  or 
inflammation.  When  the  mucus  is  small  in  amount  and 
intimately  mixed  with  the  stool,  the  trouble  is  probably 
in  the  small  intestine.  Larger  amounts,  not  well  mixed 
with  fecal  matter,  indicate  inflammation  of  the  large 
intestine.  Stools  composed  almost  wholly  of  mucus  and 
streaked  with  blood  are  the  rule  in  dysentery,  ileocoUtis, 
and  intussusception. 

In  the  so-called  mucous  colic  or  membranous  enteritis, 
shreds  and  ribbons  of  altered  mucus,  sometimes  repre- 
senting complete  casts  of  portions  of  the  bowel,  are 
passed.  These  may  appear  as  firm,  irregularly  seg- 
mented strands,  suggesting  tapeworms.  The  mucus 
sometimes  takes  the  form  of  frog-spawn-like  masses. 
In  some  cases  it  is  passed  at  variable  intervals,  with 
cohc;  in  others,  with  every  stool,  with  only  vague  pains 
and  discomfort.  It  is  distinguished  from  inflammatory 
mucus  by  absence  of  pus-corpuscles.  The  condition  is 
not  uncommon  and  should  be  more  frequently  recog- 
nized. It  is  probably  a  secretory  neurosis,  hence  the 
name  "membranous  enteritis"  is  inappropriate. 

7.  Concretions. — Gall-stones  are  probably  more  com- 
mon than  is  generally  supposed,  and  should  be  searched 
for  in  every  case  of  obscure  colicky  abdominal  pain. 
Intestinal  concretions  (enteroliths)  are  rare.  Intestinal 
sand,  consisting  of  sand-like  grains,  is  especially  common 
in  neurotic  conditions,  such  as  mucous  coUtis.  After 
ingestion  of  considerable  amounts  of  olive  oil,  nodules  of 
soap  and  fat  often  appear  in  the  feces?  ajid. may  ^be 

CULLLlilE   or    — -  - 


352  THE   FECES 

mistaken  by   the  patient   for  gall-stones,   particularly 
when  the  oil  has  been  given  for  cholelithiasis. 

Concretions  can  be  found  by  breaking  up  the  fecal 
matter  in  a  sieve  (which  may  be  improvised  from  gauze) 
while  pouring  water  over  it.  It  must  be  remembered 
that  gall-stones,  if  soft,  may  go  to  pieces  in  the  bowel. 

8.  Animal  Parasites. — Segments  of  tapeworms  and 
the  adults  and  larvae  of  other  parasites  are  often  found  in 
the  stool.  They  are  best  searched  for  in  the  manner 
described  for  concretions.  The  search  should  be  pre- 
ceded by  a  vermicide  and  a  brisk  purge.  Patients  fre- 
quently mistake  vegetable  tissue  for  intestinal  parasites, 
and  the  writer  has  known  physicians  to  make  similar 
mistakes.  Probably  the  most  frequent  sources  of  con- 
fusion are  long  fibers  from  poorly  masticated  celery  or 
"greens,"  which  suggest  round  worms;  cells  from  orange, 
which  suggest  seat  worms;  and  fibers  from  banana, 
which,  because  of  the  segmented  structure  and  the  pres- 
ence of  oval  cells,  suggest  tapeworms  and  ova.  Even 
slight  familiarity  with  the  microscopic  structure  of 
vegetable  tissue  will  prevent  the  chagrin  of  such  errors. 

9.  Curds. — The  stools  of  nursing  infants  frequently 
contain  whitish  curd-like  masses,  due  either  to  imperfect 
digestion  of  fat  or  casein  or  to  excess  of  these  in  the  diet. 
When  composed  of  fat,  the  masses  are  soluble  in  ether, 
and  give  the  Sudan  III  test.  If  composed  of  casein, 
they  will  become  tough  and  fibrous-hke  when  placed  in 
formalin  (lo  per  cent.)  for  twenty-four  hours. 

n.  CHEMIC  EXAMINATION 
Complicated  chemic  examinations  are  of  little  value  to 
the  clinician.    Certain  tests  are,  however,  important. 


CHEMIC   EXAMINATION  353 

1.  Blood. — When  present  in  large  amount  blood  pro- 
duces such  changes  in  the  appearance  of  the  stool  that 
it  is  not  Hkely  to  be  overlooked.  Traces  of  blood  (occult 
hemorrhage)  can  be  detected  only  by  special  tests. 
Recognition  of  occult  hemorrhage  has  its  greatest  value 
in  diagnosis  of  gastric  cancer  and  ulcer.  It  is  constantly 
present  in  practically  every  case  of  gastric  cancer,  and  is 
always  present,  although  usually  intermittently,  in  ulcer. 
Traces  of  blood  also  accompany  malignant  disease  of  the 
bowel,  the  presence  of  certain  intestinal  parasites,  and 
other  conditions. 

Detection  of  Occult  Hemorrhage. — Soften  a  portion  of  the 
stool  with  water,  shake  with  an  equal  volume  of  ether  to 
remove  fat,  and  discard  the  ether.  Treat  the  remaining 
material  with  about  one- third  its  volume  of  glacial  acetic 
acid  and  extract  with  ether.  Should  the  ether  not  separate 
well,  add  a  little  alcohol.  Apply  the  guaiac  or  benzidin 
test  to  the  ether  as  already  described  (see  p.  157). 

In  every  case  iron-containing  medicines  must  be  stopped, 
and  blood-pigment  must  be  excluded  from  the  food  by  giving 
an  appropriate  diet,  e.  g.,  bread,  milk,  eggs,  and  fruit.  At  the 
beginning  of  the  restricted  diet  give  a  gram  of  powdered 
charcoal  or,  better,  0.3  gram  of  carmin,  in  capsules,  so  as  to 
mark  the  corresponding  stool. 

2.  Bile. — Normally,  unaltered  bile-pigment  is  never 
present  in  the  feces  of  adults.  In  catarrhal  conditions 
of  the  small  intestine  bilirubin  may  be  carried  through 
unchanged.  It  may  be  demonstrated  by  the  test  for 
hydrobilirubin  which  follows,  or,  if  a  considerable 
amount  is  present,  by  filtering  (after  mixing  with  water 
if  the  stool  be  solid)  and  testing  the  filtrate  by  Gmehn's 
method,  as  described  under  The  Urine. 

23 


354  THE   FECES 

3.  Hydrobilirubin. — This  substance,  which  is  the  prin- 
cipal normal  pigment  of  the  feces,  is  derived  from  bili- 
rubin by  reduction  processes  in  the  intestine,  largely  the 
result  of  bacterial  activity.  It  is  apparently  identical 
with  urobilin.  HydrobiUrubin  is  absent  from  the  stool 
in  complete  obstruction  of  the  common  or  hepatic  bile- 
duct. 

Hydrobilirubin  will  give  a  red  color  if  a  Uttle  of  the 
stool  be  rubbed  up  with  saturated  mercuric  chlorid 
solution  and  allowed  to  stand  twenty-four  hours.  The 
red  color  is  likewise  imparted,  to  microscopic  structures 
which  are  stained  with  hydrobilirubin.  A  green  color 
in  this  test  shows  the  presence  of  unchanged  bilirubin. 

4.  Trypsin. — This  ferment  is  normally  present  in  the 
feces.  The  amount  is  greatest  upon  a  diet  rich  in  pro- 
teins, least  upon  a  carbohydrate  diet.  When  it  is  absent 
or  greatly  diminished,  pancreatic  insufficiency  is  to  be 
inferred.    The  test  is  usually  made  upon  a  protein  diet. 

Gross'  Test. — In  a  mortar  thoroughly  rub  up  a  portion 
of  the  fecal  mass  with  three  times  its  bulk  of  o.i  per  cent, 
sodium  carbonate  solution.  Filter.  Mix  lo  c.c.  of  the 
filtrate  with  loo  c.c.  of  a  fresh  solution  consisting  of  0.5  gram 
Griibler's  pure  casein,  i  gm.  sodium  carbonate,  and  1000  c.c. 
distilled  water.  Add  a  little  toluol  to  prevent  bacterial 
activity  and  place  in  an  incubator  at  about  38°  C.  At  inter- 
vals remove  a  few  cubic  centimeters  and  test  for  casein  by 
adding  a  few  drops  of  acetic  acid  of  about  i  per  cent,  strength. 
A  white  cloud  appears  as  long  as  any  casein  remains  undi- 
gested. With  the  patient  upon  a  protein  diet,  there  is  nor- 
mally a  sufficient  amoimt  of  trypsin  to  digest  all  the  casein 
in  ten  to  fifteen  hours.  Delay  or  complete  failure  of  diges- 
tion shows  diminution  or  absence  of  trypsin. 


MICROSCOPIC   EXAMINATION 


355 


m.  MICROSCOPIC  EXAMINATION 

Care  must  be  exercised  in  selection  of  portions  for 
examination.  A  random  search  will  often  reveal  nothing 
of  interest.  A  small  bit  of  the  stool,  or  any  suspicious- 
looking  particle,  is  placed  upon  a  sHde,  softened  with 
water  if  necessary,  and  pressed  out  into  a  thin  layer  with 
a  cover-glass.  A  large  slide — about  2  by  3  inches — 
with  a  correspondingly  large  cover  will  be  iound  conve- 


=^:-:ir---3. 


Fig.  log. — Microscopic  elements  of  normal  feces:  a,  Muscle-fibers;  b,  connective  tissue; 
c,  epithelial  cells;  d,  white  blood -corpuscles;  e,  spiral  vessels  of  plants; /-A,  vegetable  cells; 
«,  plant  hairs;  k,  triple  phosphate  crystals;  /,  stone  cells.  Scattered  among  these  elements 
are  micro-organisms  and  debris  (after  v.  Jaksch). 


nient.  Most  of  the  structures  which  it  is  desired  to  see 
can  be  found  with  a  i6-mm.  objective.  Details  of  struc- 
ture must  be  studied  with  a  higher  power. 

The  bulk  of  the  stool  consists  of  granular  debris. 
Among  the  recognizable  structures  met  in  normal  and 
pathologic  conditions  are:  Remnants  of  food,  epitheUal 
cells,  pus-corpuscles,  red  blood-corpuscles,  crystals,  bac- 
teria, and  ova  of  animal  parasites  (Fig.  109). 


356  THE   FECES 

1.  Remnants  of  Food.— These  include  a  great  variety 
of  structures  which  are  very  confusing  to  the  student. 
Considerable  study  of  normal  feces  is  necessary  for  their 
recognition. 

Vegetable  fibers  are  generally  recognized  from  their 
spiral  structure  or  their  pits,  dots,  or  reticulate  mark- 
ings; vegetable  cells,  from  their  double  contour  and  the 
chlorophyl  bodies  which  many  of  them  contain.  These 
cells  are  apt  to  be  mistaken  for  the  ova  of  parasites. 


/ 


Fig.  no. — Poorly  digested  muscle-fiber  in  feces  showing  striations  (X200)  (photograph 
by  the  author). 

Vegetable  hairs  (Fig.  109,  i)  frequently  look  much  like 
the  larvae  of  some  of  the  worms.  Anything  like  a  careful 
examination  will,  however,  easily  distinguish  them, 
because  of  the  homogeneous  and  highly  refractile  wall, 
the  distinct  central  canal  which  extends  the  whole 
length,  and,  especially,  the  absence  of  motion.  Starch- 
granules  sometimes  retain  their  original  form,  but  are 
ordinarily  not  to  be  recognized  except  by  their  staining 
reaction.    They  strike  a  blue  color  with  Lugol's  solu- 


MICROSCOPIC   EXAMINATION  357 

tion  when  undigested;  a  red  color,  when  slightly  digested. 
Muscle-fibers  are  yellow,  and  when  poorly  digested  appear 
as  short,  transversely  striated  cylinders  with  rather 
squarely  broken  ends  (Fig.  no).  Generally,  the  ends 
are  rounded  and  the  striations  faint,  or  only  irregularly 
round  or  oval  yellow  masses  are  found.  Curds  of  milk  are 
especially  important  in  the  stools  of  children.  They  must 
be  distinguished  from  small  masses  of  fat  (see  p.  352). 

Excess  of  any  of  these  structures  may  result  from 
excessive  ingestion  or  deficient  intestinal  digestion. 

2.  Epithelial  Cells. — A  few  cells  derived  from  the 
wall  of  the  alimentary  canal  are  a  constant  finding.  They 
show  all  stages  of  degeneration,  and  are  often  unrecog- 
nizable. A  marked  excess  has  its  origin  in  a  catarrhal 
condition  of  some  part  of  the  bowel.  Squamous  cells 
come  from  the  anal  orifice;  otherwise  the  form  of  the 
cells  gives  no  clue  to  the  location  of  the  lesion. 

3.  Pus. — ^Amounts  of  pus  sufiicient  to  be  recognized 
with  the  eye  alone  indicate  rupture  of  an  abscess  into 
the  bowel.  If  well  mixed  with  the  stool,  the  source  is 
high  up,  but  in  such  cases  the  pus  is  apt  to  be  more 
or  less  completely  digested,  and  hence  unrecognizable. 
Small  amounts,  detected  only  by  the  microscope,  are 
present  in  catarrhal  and  ulcerative  conditions  of  the  in- 
testine, the  number  of  pus-cells  corresponding  to  the 
severity  and  extent  of  the  process. 

4.  Blood=corpuscles. — Unaltered  red  corpuscles  are 
rarely  found  unless  their  source  is  near  the  anus.  Ordi- 
narily, only  masses  of  blood-pigment  can  be  seen.  Blood 
is  best  recognized  by  the  chemic  tests  (see  p.  353). 

5.  Bacteria. — In  health,  bacteria— mostly  dead — con- 
stitute about  one-third  of  the  weight  of  the  dried  stool. 


358  THE   FECES 

They  are  beneficial  to  the  organism,  although  not 
actually  necessary  to  its  existence.  Under  certain  con- 
ditions they  may  be  harmful.  It  is  both  difficult  and 
unprofitable  to  identify  them.  The  great  majority 
belong  to  the  colon  bacillus  group,  and  are  negative  to 
Gram's  method  of  staining. 

In  some  pathologic  conditions  the  character  of  the 
intestinal  flora  changes,  so  that  Gram-staining  bacteria 
very  greatly  predominate.  As  shown  by  R.  Schmidt, 
of  Neusser's  clinic  in  Vienna,  this  change  is  most  con- 
stant and  most  striking  in  cancer  of  the  stomach,  owing 
to  large  numbers  of  Boas-Oppler  bacilli,  and  is  of  con- 
siderable value  in  diagnosis.  He  believes  that  a  diagnosis 
of  gastric  carcinoma  should  be  very  unwillingly  made 
with  an  exclusively  "Gram-negative"  stool,  while  a 
"Gram -positive"  stool,  due  to  bacilli  (which  should  also 
stain  brown  with  Lugol's  solution),  may  be  taken  as  very 
strong  evidence  of  cancer.  A  Gram-positive  stool  due 
to  cocci  is  suggestive  of  intestinal  ulceration.  The  tech- 
nic  is  the  same  as  when  Gram's  method  is  applied  to 
other  material  (see  p.  467),  except  that  the  smear  is  fixed 
by  immersion  in  methyl-alcohol  for  five  minutes  instead 
of  by  heat.  Pyronin  is  a  good  counterstain.  The  deep 
purple  Gram-staining  bacteria  stand  out  more  prom- 
inently than  the  pale-red  Gram-negative  organisms,  and 
one  may  be  misled  into  thinking  them  more  numerous 
even  in  cases  in  which  they  are  much  in  the  minority. 
The  number  of  Boas-Oppler  bacilli  can  be  increased  by 
administering  a  few  ounces  of  sugar  of  milk  the  day 
before  the  examination.  The  bacteria  can  be  obtained 
comparatively  free  from  food  remnants  by  mixing  a 
little  of  the  feces  with  water,  allowing  to  settle  for  a 


MICROSCOPIC   EXAMINATION  359 

short  time,  and  making  smears  from  the  supernatant 
fluid. 

Owing  to  the  diflSculty  of  excluding  swallowed  sputum, 
the  presence  of  the  tubercle  bacillus  is  less  significant  in 
the  feces  than  in  other  material.  It  may,  however,  be 
taken  as  evidence  of  intestinal  tuberculosis  when  clinical 
signs  indicate  an  intestinal  lesion  and  reasonable  care  is 
exercised  in  regard  to  the  sputum.  Success  in  the 
search  will  depend  largely  upon  careful  selection  of  the 
portion  examined.  A  random  search  will  almost  surely 
fail.  Whitish  or  grayish  flakes  of  mucus  or  blood- 
stained or  purulent  particles  should  be  spread  upon 
slides  or  covers  and  stained  by  the  method  given  upon  p. 
205.  In  the  case  of  rectal  ulcers,  swabs  can  be  made 
directly  from  the  ulcerated  surface.  With  young  chil- 
dren who  swallow  all  their  sputum  an  examination  of 
the  stool  for  tubercle  bacilli  may  be  the  means  of  diag- 
nosing tuberculosis  of  the  lung. 

6.  Crystals. — Various  crystals  may  be  found,  but  few 
have  any  significance.  Slender,  needle-like  crystals  of 
fatty  acids  and  soaps  (see  Fig.  37)  and  triple  phosphate 
crystals  (see  Fig.  109)  are  common.  Characteristic  oc- 
tahedral crystals  of  calcium  oxalate  (see  Fig.  52)  appear 
after  ingestion  of  certain  vegetables.  Charcot-Leyden 
crystals  (see  Fig.  9)  are  not  infrequently  encountered, 
and  strongly  suggest  the  presence  of  intestinal  parasites. 
Yellowish  or  brown,  needle-like  or  rhombic  crystals  of 
hematoidin  (see  Fig.  37)  may  be  seen  after  hemorrhage 
into  the  bowel.  The  dark  color  of  the  feces  after  ad- 
ministration of  bismuth  salts  is  due  largely  to  great 
numbers  of  bismuth  suboxid  crystals.  They  resemble 
hemin  crystals. 


360  THE   FECES 

7.  Parasites  and  Ova. — Descriptions  will  be  found 
in  the  following  chapter.  The  flagellates  are  usually 
best  found  in  the  second  stool  after  a  saline  cathartic, 
the  first  stool  being  ordinarily  too  solid  and  the  later 
ones  too  dilute. 

To  find  ova  when  scarce,  they  must  be  concentrated. 
Stiles  advises  thoroughly  mixing  the  stool  with  a  quart 
or  more  of  water,  allowing  to  settle,  pouring  off  the  water 
almost  down  to  the  sediment,  and  repeating  the  process 
as  long  as  any  matter  floats.  The  final  sediment  is 
poured  into  a  conical  glass  and  allowed  to  settle.  Ova 
will  be  found  in  the  fine  sediment,  which  can  readily 
be  removed  with  a  pipet.  Other  and  more  complicated 
methods  have  been  devised,  but  this  and  Pepper's 
method  for  hookworm  eggs  (see  p.  413)  will  probably 
answer  all  clinical  needs. 

IV.  FUNCTIONAL  TESTS 

1.  Schmidt's  Test  Diet. — Much  can  be  learned  of  the 
various  digestive  functions  from  a  microscopic  study  of 
the  feces,  especially  when  the  patient  is  upon  a  known 
diet.  For  this  purpose  the  standard  diet  of  Schmidt  is 
generally  adopted.     This  consists  of: 

Morning 0.5  liter  milk  and  50  gm.  toast. 

Forenoon 0.5  liter   porridge,  made  as  follows:  40  gm. 

oatmeal,  10  gm.  butter,  200  c.c.  milk, 
300  c.c.  water,  one  egg,  and  salt  to  taste. 

Midday 125  gm.  hashed  meat,  with  20  gm.  butter, 

fried  so  that  the  interior  is  quite  rare; 
250  gm.  potato,  made  by  cooking  190 
gm.  potato  with  100  c.c.  milk  and  10  gm. 
butter,  the  whole  boiled  down  to  250  c.c. 

Afternoon Same  as  morning. 

Evening Same  as  forenoon. 


FUNCTIONAL   TESTS  361 

At  the  beginning  of  the  diet,  the  stool  should  be 
marked  off  with  carmin  or  charcoal  (see  p.  362).  One 
should  familiarize  himself  with  the  microscopic  appear- 
ance of  the  feces  of  normal  persons  upon  this  diet. 

Deficiency  of  starch  digestion  is  recognized  by  the 
number  of  starch-granules  which  strike  a  blue  color  with 
iodin.  With  exception  of  those  inclosed  in  plant  cells 
none  are  present  normally. 

The  degree  of  protein  digestion  is  ascertained  by  the 
appearance  of  the  muscle-fibers.  Striations  are  clearly 
visible  on  any  considerable  number  of  the  fibers  only 
when  digestion  is  imperfect  (see  Fig.  no).  They  usually 
disappear  after  the  feces  have  stood  for  some  time. 
According  to  Schmidt,  the  presence  of  nuclei  in  muscle- 
fibers  denotes  complete  absence  of  pancreatic  function. 
The  presence  of  connective-tissue  shreds  indicates  de- 
ficient gastric  digestion,  since  raw  connective  tissue  is 
digested  only  in  the  stomach.  These  shreds  can  be 
recognized  macroscopically  by  examining  in  a  thin  layer 
against  a  black  background,  and  microscopically  by 
their  fibrous  structure  and  the  fact  that  they  clear  up 
when  treated  with  acetic  acid.  Digestion  of  fats  is 
checked  up  by  the  amount  of  neutral  fat. 

Schmidt's  nuclei  test  for  pancreatic  insufficiency  con- 
sists in  the  administration  of  a  |-cm.  cube  of  fresh  beef 
tied  in  a  little  gauze  bag  with  the  test-meal.  When  the 
bag  appears  in  the  feces  it  is  opened  and  its  contents 
examined  microscopically  by  pressing  out  small  bits 
between  a  slide  and  cover.  A  drop  of  some  nuclear 
-  stain  may  be  applied  if  desired.  If  the  nuclei  are  for  the 
most  part  undigested,  pancreatic  insufficiency  may  be 
assumed,  since  it  is  probable  that  nuclei  can  be  digested 


362  THE   FECES 

only  by  the  pancreatic  juice.  Normally  the  nuclei  are 
digested,  provided  the  time  of  passage  through  the 
intestine  is  not  less  than  six  hours. 

2.  Sahli's  Qlutoid  Test.— The  Schmidt  test  diet  in- 
volves some  inconvenience  for  the  patient,  and  inter- 
pretation of  results  requires  much  experience  upon  the 
part  of  the  physician.  A  number  of  other  methods  of 
testing  the  digestive  functions  have  been  proposed.  The 
glutoid  test  of  Sahli  is  one  of  the  most  satisfactory. 
This  is  similar  to  his  desmoid  test  of  gastric  digestion 
described  on  page  346.  A  glutoid  capsdle  containing 
0.15  gram  iodoform  is  taken  with  an  Ewald  breakfast. 
The  capsule  is  not  digested  by  the  stomach  fluid,  but 
is  readily  digested  by  pancreatic  juice.  Appearance  of 
iodin  in  the  saliva  and  urine  within  four  to  six  hours 
indicates  normal  gastric  motility,  normal  intestinal  di- 
gestion, and  normal  absorption.  Instead  of  iodoform, 
0.5  gram  salol  may  be  used,  salicyluric  acid  appearing 
in  the  urine  in  about  the  same  time.  For  tests  for  iodin 
and  salicyluric  acid,  see  pages  346  and  347. 

Glutoid  capsules  are  prepared  by  soaking  gelatin  cap- 
sules in  formalin.  Sahli  states  that  filled  capsules  can  be 
purchased  of  A.  G.  Haussmann,  in  St.  Gall,  Switzerland. 

3.  Motility. — Ordinarily,  fifteen  to  thirty  hours  are 
required  for  the  passage  of  ingested  material  through 
the  gastro-intestinal  tract.  In  diarrheal  conditions  the 
time  is  usually  much  shortened,  unless  the  pathologic 
process  is  in  the  colon.  In  intestinal  stasis  it  may  be 
much  prolonged.  The  time  of  passage  is  acertained  by 
giving  0.5  gm.  of  powdered  charcoal  or  0.3  gm.  of  car- 
mine in  a  capsule  with  a  meal  and  watching  for  the 
resulting  discolored  feces. 


CHAPTER  VI 

ANIMAL  PARASITES 

Animal  parasites  are  common  in  all  countries,  but  are 
especially  abundant  in  the  tropics,  where,  in  some  locali- 
ties, almost  every  native  is  host  for  one  or  more  species. 
Because  of  our  growing  intercourse  with  these  regions 
the  subject  is  assuming  increasing  importance  in  this 
country.  Many  parasites,  hitherto  comparatively  un- 
known here,  will  probably  become  fairly  common. 

Some  parasites  produce  no  symptoms,  even  when 
present  in  large  numbers.  Others  cause  very  serious 
symptoms.  It  is,  however,  impossible  to  make  a  sharp 
distinction  between  pathogenic  and  non-pathogenic 
varieties.  Parasites  which  cause  no  apparent  ill  effects 
in  one  individual  may,  under  certain  conditions,  produce 
marked  disturbances  in  another.  The  disturbances  are 
so  varied,  and  frequently  so  indefinite,  that  diagnosis  can 
rarely  be  made  from  the  clinical  symptoms.  It  must  rest 
upon  detection,  by  the  naked  eye  or  the  microscope,  of 
(a)  the  parasites  themselves,  (b)  their  ova  or  larvae,  or 
(c)  some  of  their  products. 

Unlike  bacteria,  the  great  majority  of  animal  parasites 
multiply  by  means  of  alternating  and  differently  formed 
generations,  which  require  widely  different  conditions 
for  their  development.  The  few  exceptions  are  chiefly 
-among  the  protozoa.  Multiplication  of  parasites  within 
the  same  host  is  thus  prevented.     In  the  case  of  the 

363 


364  ANIMAL   PARASITES 

hookworm,  for  example,  there  is  no  increase  in  the  num- 
ber of  worms  in  the  host's  intestine,  except  through  re- 
infection from  the  outside.  The  ova  are  carried  out  of 
the  intestine  and  the  young  must  pass  a  certain  period 
of  development  in  warm,  moist  earth  before  they  can 
again  enter  the  human  body  and  grow  to  maturity. 
This  also  explains  the  geographic  distribution  of  para- 
sites. The  hookworm  cannot  flourish  in  cold  countries; 
malaria  can  prevail  only  in  localities  in  which  the  mos- 
quito, Anopheles,  exists,  and  then  only  after  the  mos- 
quitoes have  become  infected  from  a  human  being. 

In  general,  this  alternation  of  periods  of  development 
takes  place  in  one  of  three  ways: 

(i)  The  young  remain  within  the  original  host,  but 
travel  to  other  organs,  where  they  do  not  reach  maturity, 
but  lie  quiescent  until  taken  in  by  a  new  host.  A  good 
example  is  Trichinella  spiralis. 

(2)  The  young  or  the  ova  which  subsequently  hatch 
pass  out  of  the  host,  and  either  {a)  go  through  a  simple 
process  of  growth  and  development  before  entering 
another  host,  as  is  the  case  with  the  hookworm,  or  {h) 
pass  through  one  or  more  free-living  generations,  the 
progeny  of  which  infect  new  hosts,  as  is  the  case  with 
Strongyloides  intestinalis. 

(3)  The  young  or  ova  or  certain  specialized  forms 
either  directly  (e.  g.,  malarial  parasites)  or  indirectly 
(e.  g.,  tapeworms)  reach  a  second  host  of  different 
species,  where  a  widely  different  process  of  develop- 
ment occurs.  The  host  in  which  the  adult  or  sexual 
existence  is  passed  is  called  the  definitive  or  final  host; 
that  in  which  the  intermediate  or  larval  stage  occurs,  the 
intermediate  host.     Man,  for  example,  is  the  definitive 


ANIMAL   PARASITES  365 

host  for  TcBfiia  saginata,  and  the  intermediate  host  for 
the  malarial  parasites  and  Tcenia  echinococcus. 

A  few  words  concerning  the  classification  and  nomen- 
clature of  living  organisms  in  general  will  be  helpful 
at  this  place.  Individuals  which  are  alike  in  all  essential 
respects  are  classed  together  as  a  species.  Closely  related 
species  are  grouped  together  to  form  a  genus;  genera, 
which  have  certain  characteristics  in  common  make  up 
a  family;  families  are  grouped  into  orders;  orders,  into 
classes;  and  classes,  finally,  into  the  branches  or  phyla, 
which  make  up  the  animal  and  vegetable  kingdoms.  In 
some  cases  these  groups  are  subdivided  into  intermedi- 
ate groups — subphyla,  subfamiHes,  etc.,  and  occasionally 
shght  differences  warrant  subdivision  of  the  species  into 
varieties. 

The  scientific  name  of  an  animal  or  plant  consists  of 
two  parts,  both  Latin  or  Latinized  words,  and  is  printed 
in  italics.  The  first  part  is  the  name  of  the  genus  and 
begins  with  a  capital  letter;  the  second  is  the  name  of  the 
species  and  begins  with  a  lower  case  letter,  even  when  it 
was  originally  a  proper  name.  When  there  are  varieties 
of  a  species,  a  third  part,  the  designation  of  the  variety, 
is  appended.  The  author  of  the  name  is  sometimes  in- 
dicated in  Roman  type  immediately  after  the  name  of 
the  species.  Examples:  Spirochata  vincenti,  often  abbre- 
viated to  Sp.  vincenti  when  the  genus  name  has  been 
used  just  previously;  Staphylococcus  pyogenes  alhus; 
Necator  americanus,  Stiles. 

At  the  present  time  there  is  great  confusion  in  the 
naming  and  classification  of  parasites.  Some  have  been 
given  a  very  large  number  of  names  by  different  ob- 
servers, and  in  many  cases  different  parasites  have  been 


366  ANIMAL   PARASITES 

described  under  the  same  name.  The  alternation  of 
generations  and  the  marked  differences  in  some  cases 
between  male  and  female  have  contributed  to  the  con- 
fusion, different  forms  of  the  same  parasite  being  de- 
scribed as  totally  unrelated  species. 

The  number  of  parasites  which  have  been  described 
as  occurring  in  man  and  the  animals  is  extremely  large. 
Only  those  which  are  of  medical  interest  are  mentioned 
here.  They  belong  to  four  phyla — Protozoa,  Platyhel- 
minthes,  Nemathelminthes,  and  Arthropoda. 

PHYLUM  PROTOZOA 

These  are  unicellular  organisms,  the  simplest  types 
of  animal  life.  There  is  very  Uttle  differentiation  of 
structure.  Each  contains  at  least  one,  and  some  several, 
nuclei.  Some  contain  contractile  vacuoles;  some  have 
cilia  or  flagella  as  special  organs  of  locomotion.  They 
reproduce  by  division,  by  budding,  or  by  sporulation. 
Sometimes  there  is  an  alternation  of  generations,  in  one 
of  which  sexual  processes  appear,  as  is  the  case  with  the 
malarial  parasites.  The  protozoa  are  very  numerous, 
the  subphylum  Sarcodina  alone  including  no  less  than 
5000  species.  Most  of  the  protozoa  are  microscopic  in 
size;  a  few  are  barely  visible  to  the  naked  eye.  The 
beginning  student  can  gain  a  general  idea  of  their  appear- 
ance by  examining  water  (together  with  a  Uttle  of  the 
sediment)  from  the  bottom  of  any  pond.  Such  water 
usually  contains  amebae  and  a  considerable  variety  of 
ciliated  and  flagellated  forms. 

The  following  is  an  outline  of  those  protozoa  which 
are  of  medical  interest,  together  with  the  subphyla  and 
classes  to  which  they  belong: 


PHYLUM   PROTOZOA 


367 


PHYLUM  PROTOZOA 

SuBPHYLUM   I.     SARCODINA. — Locomotion  by  means  of  pseudo- 
podia. 
Class  Rhizopoda. — Pseudopodia  form  lobose  or  reticulose  processes. 


Genus. 
Entamoeba. 


Species. 
E.  histolytica. 
E.  coli. 
E.  buccalis. 


Stjbphylum  II.  MASTIGOPHORA  (FLAGELLATA).— Locomotion 
by  means  of  flagella. 

Class  Zodmastigophora. — Forms  in  which  animal  characteristics  pre- 
dominate. 


Genus. 

Species. 

Spirochaeta. 

Sp.  recurrentis. 

Sp.  vincenti. 

Sp.  buccalis. 

Sp.  dentium. 

Sp.  refringens. 

Treponema. 

T.  pallidum. 

» 

T.  pertenue. 

Trypanosoma. 

T.  gambiense. 

T.  rhodesiense. 

T.  cruzi. 

T.  lewisi. 

T.  evansi. 

T.  brucei. 

T.  equiperdum. 

Leishmania. 

L.  donovani. 

L.  tropica. 

L.  infantum. 

Cercomonas. 

C.  hominis. 

Bodo. 

B.  urinarius. 

Trichomonas. 

T.  vaginalis. 

T.  intestinalis. 

T.  pulmonalis. 

Lamblia. 

L.  intestinalis. 

368  ANIMAL    PARASITES 

SuBPHYLUM  III.    SPOROZOA. — All    members   parasitic.    Propaga- 
tion by  means  of  spores.    No  special  organs  of  locomotion. 

Class  Telosporidia. — Sporulation  ends  the  life  of  the  individual. 

Genus.  Species. 

Coccidium.  C.  cuniculi. 

Plasmodium.  P.  viva.\. 

P.  malariae. 

P.  falcipanun. 

P.  tenue. 
Babesia.  B.  bigeminum. 

SuBPHYLUM  IV.    INFUSORIA, — Locomotion  by  means  of  dlia. 
Class  Ciliata. — Cilia  present  throughout  life. 

Genus.  Species. 

Balantidium.  B.  coli. 

SUBPHYLUM   SARCODINA 
Class  Rhizopoda 

These  are  protozoa  the  body  substance  of  which 
forms  changeable  protoplasmic  processes,  or  pseudo- 
podia,  for  the  taking  in  of  food  and  for  locomotion. 
They  possess  one  or  several  nuclei. 

I.  Genus  Entamoeba. — (i)  Entamoeba  histolytica. — 
This  organism  is  found,  often  in  large  numbers,  in  the 
stools  of  tropical  dysentery  and  in  the  pus  and  walls 
of  hepatic  abscesses  associated  with  dysentery.  Infec- 
tion is  more  common  in  this  country  than  is  usually 
supposed.  It  is  a  grayish  or  colorless,  granular  cell, 
usually  between  25  and  40  ;f^  in  diameter  (Fig.  iii). 
Its  appearance  varies  according  to  its  stage  of  develop- 
ment. In  the  vegetative  stage,  which  is  found  in  acute 
dysentery,  there  is  a  distinct  homogeneous,  refractile 
ectoplasm  and  a  granular  endoplasm  containing  one  or 
more  distinct  vacuoles,  a  round  nucleus  which  is  ordi- 
narily very  indistinct,  and  frequently  ingested  red  blood- 


PHYLUM   PROTOZOA 


369 


corpuscles  and  bacteria.  When  at  rest  its  shape  is 
spheric,  but  upon  a  warm  slide  it  exhibits  the  charac- 
teristic ameboid  motion,  constantly  changing  its  shape 
or  moving  actively  about  by  means  of  distinct  pseudo- 
podia.  This  motion  is  its  most  distinctive  feature, 
and  should  always  be  seen  to  estabhsh  the  identity  of 
the  organism  in  this  stage.  It  is  lost  when  the  speci- 
men cools,  and  can  usually  not  be  re-estabHshed  by 


Fig.  III. — Entamoeba  histolytica  in  intestinal  mucus,  with  blood-corpuscles  and  bacteria 

(Losch). 


warming.  According  to  Simon,  if  neutral  red  in  0.5 
per  cent,  solution  be  run  under  the  cover-glass,  it  will 
be  taken  up  by  the  entamebae  and  other  protozoa  and 
will  render  them  conspicuous  without  killing  them 
("vital  staining").  Pauton  recommends  that  the  sus- 
pected material  be  mounted  in  a  drop  of  aqueous 
methylene-blue  solution  and  covered.  Body  cells  will 
be  stained  blue,  while  the  entamebae  remain  colorless 
and  stand  out  distinctly  against  a  blue  background. 

24 


37°  ANIMAL    PARASITES 

In  dysentery  "carriers"  and  in  chronic  cases  when 
the  stools  are  formed  and  hard  most  or  all  of  the  para- 
sites may  become  encysted.  Their  appearance  in  this 
stage  of  development  is  given  in  the  table  on  pages 

371,372- 

When  the  presence  of  entamebae  is  suspected,  the 
stool  should  be  passed  into  a  warm  vessel  and  kept 
warm  until  and  during  the  examination.  A  warm  stage 
can  be  improvised  from  a  plate  of  copper  with  a  hole 
cut  in  the  center.  This  is  placed  upon  the  stage  of  the 
microscope,  and  one  of  the  projecting  ends  is  heated 
with  a  small  flame.  Entamebae  are  most  likely  to  be 
found  in  grayish  or  blood-streaked  particles  of  mucus. 
Craig  recommends  the  liquid  stool  following  a  saline 
cathartic.  Favorable  material  for  examination  can 
often  be  obtained  at  one's  convenience  by  inserting  into 
the  rectum  a  large  catheter  with  roughly  cut  lateral 
openings.  A  sufficient  amount  of  mucus  or  fecal  matter 
will  usually  be  brought  away  by  it. 

No  staining  method  is  as  useful  in  diagnosis  as  the 
study  of  the  li\dng  parasite.  For  more  scientific  study 
Darling  recommends  the  following  method:  Stain  with 
Wright's  (or  Hasting's  or  Leishman's)  stain  in  the 
usual  way,  and  follow  this  wath  Giemsa's  stain  imtil 
the  film  has  a  purple  cast.  Then  plunge  the  prepara- 
tion into  a  small  beaker  of  60  per  cent,  alcohol  to  which 
10  to  20  drops  of  ammonia  have  been  added  and  keep 
it  in  motion  until  the  desired  differentiation  is  obtained, 
when  the  film  will  have  a  violet  color. 

(2)  Other  Entamebae. — Entamceha  coli,  a  similar  but 
somewhat  smaller  organism,  rarely  over  25  (J.  in  diam- 
eter, has  frequently  been  found  in  the  stools  of  healthy 


PHYLUM   PROTOZOA  37 1 

persons.  It  has  less  distinct  pseudopodia,  less  sharp 
differentiation  between  ectoplasm  and  endoplasm,  less 
active  motion  and  more  distinct  nucleus,  and  does  not 
contain  ingested  red  corpuscles  or  never  more  than  one 
or  two. 

The  principal  points  of  distinction  between  E.  histo- 
lytica and  E.  coli  are  included  in  the  following  table 
which  is  slightly  modified  from  Craig^: 

VEGETATIVE   STAGE 
This  stage  of  E.  histolytica  is  found  in  acute  dysentery. 

Entamoeba  histolytica.  Entamosba  coli. 

Averages  larger.    Unimportant.         Averages  smaller. 

Actively  motile.      Characterls-         Sluggishly       motile.       Seldom 
tic.     Often  moves  from  place  to     moves  from  place  to  place, 
place. 

Ectoplasm    hyaline,    glass-like,         Ectoplasm  not  glass-like,  poorly 
sharply  differentiated  from  endo-     differentiated  from  endoplasm. 
plasm.     Characteristic. 

Nucleus  usually  indistinct,  often         Nucleus  distinct.     Located  near 
invisible.     Changes  position  with     center, 
motion  of  parasite. 

Red  blood-cells  present  in  endo-  No  red  blood-cells  (or  never 
plasm  when  stool  contains  blood,  more  than  one  or  two)  in  endo- 
Very  characteristic.  plasm  when  stool  contains  blood. 

PRECYSTIC   STAGE 

E.  histolytica  may  be  found  in  this  stage  when  symptoms 
of  dysentery  have  practically  disappeared.  The  parasite  is 
reduced  in  size,  is  sluggishly  motile,  and  becomes  practically 
indistinguishable  from  E.  coli.  The  distinction  must  be 
based  upon  the  vegetative  or  cystic  forms,  a  few  of  which 
can  usually  be  found  in  the  same  stool. 

'  Craig:  Archives  of  Internal  Medicine,  1914,  xiii,  917. 


372  ANIMAL   PARASITES 

CYSTIC  STAGE 

In  formed  stools  both  entamebae  are  commonly  encysted. 
This,  therefore,  is  the  form  of  E.  histolytica  to  be  looked  for 
between  recurrences  and  in  dysentery  "carriers." 

Entamoeba  histolylica.  Enlamoeba  coli. 

Cysts  spheric   or   oval.      Cyst         Similar,  but  double  outline  of 
wall  single  and  delicate  in  young     wall    more    frequently    observed 
cysts;      thicker     and     sometimes     and  more  distinct, 
double  outlined  in  older  ones. 

Diameter  10-20  /';  average,  12  /i.         Diameter  10-25 ,";  average,  15  fi. 

Cytoplasm  of  young  cysts  gran-         Similar,    but    chromidia    very 
ular,  often  with  a  large  vacuole,     rare. 
Pressure    of    chromidia    (brightly 
refractive,    spindle-shaped    or    ir- 
regular masses  of  chromatin)  char- 
acteristic. 

Fully  developed   cysts   contain         Fully  developed   cyst   contains 
four  distinct  nuclei  seen  by  focus-     eight  to  sixteen  nuclei,  eight  being 
ing  at  different  levels.    Very  char-     the  normal  number, 
acteristic. 

E.  tetragena,  which  was  described  in  1907  by  Viereck,  is 
now  regarded  as  identical  with  E.  histolytica.  Another, 
E.  buccaUs,  has  been  found  in  decaying  teeth.  A  num- 
ber of  similar  organisms  have  been  described  as  occurring 
in  pus  and  in  ascitic  and  other  body  fluids,  but  it  is 
probable  that  in  many  cases,  at  least,  the  structures 
seen  were  ameboid  body  cells. 

SUBPHYLUM   MASTIGOPHORA    (FLAGELLATA) 

Class  Zobmastigophora 

The  protozoa  of  this  subphylum  are  provided  with  one 

or  several  whip-like  appendages  with  lashing  motion, 

termed   flagella,   which   serve   for   locomotion   and,   in 

some  cases,  for  feeding.    They  generally  arise  from  the 


PHYLUM   PROTOZOA  373 

anterior  part  of  the  organism.  Some  members  of  the 
group  also  possess  an  undulating  membrane — a  delicate 
membranous  fold  which  extends  the  length  of  the  body, 
and  somewhat  suggests  a  fin.  When  in  active  motion 
this  gives  the  impression  of  a  row  of  cilia.  The  flagellata 
do  not  exhibit  ameboid  motion,  and,  in  general,  maintain 
an  unchanging  oval  or  spindle  shape,  and  contain  a  single 
nucleus.  The  cytoplasm  contains  numerous  granules 
and  usually  several  vacuoles,  one  or  more  of  which  may 
be  contractile.  Encystment  as  a  means  of  resisting 
unfavorable  conditions  is  common, 

1.  Genus  Spirochaeta. — The  spirochetes  appear  to 
occupy  a  position  midway  between  the  bacteria .  and 
protozoa,  but  are  more  frequently  described  with  the 
latter. 

(i)  Spirochaeta  recurrentis. — This  spirochete  was 
described  by  Obermeier  as  the  cause  of  relapsing  fever. 
It  appears  in  the  circulating  blood  during  the  febrile 
attack,  and,  unlike  the  malarial  parasite,  lives  in  the 
plasma  without  attacking  the  red  corpuscles.  The 
organism  is  an  actively  motile  spiral,  1 6  to  40  u  long, 
with  three  to  twelve  wide,  fairly  regular  turns.  It  can 
be  seen  in  fresh  unstained  blood  with  a  high  dry  lens, 
being  located  by  the  commotion  which  it  creates  among 
the  red  cells.  For  diagnosis,  thin  films,  stained  with 
Wright's  or  some  similar  blood-stain,  are  used  (Fig.  112). 
In  such  preparations  the  spirals  are  not  so  regular. 

Besides  Spirochaeta  recurrentis,  a  number  of  distinct 
strains  have  been  described  in  connection  with  different 
types  of  relapsing  fever:  Sp.  novyi  (Plate  VII),  Sp.  kochi, 
Sp.  duttoni,  and  Sp.  carteri. 

(2)  Spirochaeta  vincenti. — In  stained  smears  from  the 


374 


ANIMAL   PARASITES 


ulcers  of  Vincent's  angina  (see  p.  436)  are  found  what 
appear  to  be  two  organisms.  One,  the  "fusiform  bacil- 
lus," is  a  slender  rod,  6  to  12  /u  long,  pointed  at  both 
ends  and  sometimes  curved.  The  other  is  a  slender 
spiral  organism,  30  to  40  u  long,  with  three  to  eleven 
comparatively  shallow  turns  (see  Fig.  161).  These  were 
formerly  thought  to  be  bacteria,  a  spirillum  and  bacillus 
Uving  in  symbiosis.    The  present  tendency  is  to  regard 


Fig.  112. — Spirochete  of  relapsing  fever  in  blood  (Xiooo)  (Karg  and  Schraorl). 

them  as  stages  or  forms  of  the  same  organism,  and  to 
class  them  among  the  spirochetes.  The  same  organisms 
are  quite  constantly  present  in  large  numbers  in  ulcera- 
tive stomatitis  and  in  noma.  They  are  not  infrequently 
found  in  small  numbers  in  normal  mouths. 

(3)  Other  Spirochetes. — A  number  of  harmless  forms 
are  of  interest  because  of  the  possibility  of  confusing 
them  with  the  more  important  pathogenic  varieties. 
Of  these,  Sp.  buccalis  and  Sp.  dentium  are  inhabitants 


PHYLUM   PROTOZOA  375 

of  the  normal  mouth.  The  former  is  similar  in  morphol- 
ogy to  Sp.  vincenti.  Sp.  dentium  (Fig.  113)  is  smaller, 
more  delicate,  has  deep  curves,  and  may  be  easily  mis- 
taken for  Treponema  pallidum.  It,  also,  stains  reddish 
with  Giemsa's  stain.  In  suspected  syphilitic  sores  of 
the  mouth  it  is,  therefore,  important  to  make  smears 
from  the  tissue  juices  rather  than  from  the  surface 
(see  p.  446).  Thibaudeau  has  found  it  or  a  similar  or- 
ganism in  as  high  as  41  per  cent,  of  normal  mouths. 
Sp.  refringens  is  frequently  present  upon  the  surface 


A  B  C 

Fig.   113. — Spiral  organisms:  A,  Treponema  pallidum;  B,  Spirochaeta  refringens;   C, 
Spirochaeta  dentium.    Two  red  corpuscles  are  also  shown  (X1200). 

of  ulcers,  especially  about  the  genitals,  and  has  doubtless 
many  times  been  mistaken  for  Treponema  pallidum.  It 
can  be  avoided  by  properly  securing  the  material  for 
examination;  but  its  morphology  should  be  sufficient  to 
prevent  confusion.  It  is  thicker  than  the  organism  of 
syphilis,  stains  more  deeply,  and  has  fewer  and  shal- 
lower curves  (Fig.  113).  Giemsa's  stain  gives  it  a  bluish 
color. 

2.  Genus  Treponema. — (i)  Treponema  pallidum. — 
This  is  the  organism  of  syphilis.  Its  description  and 
methods  of  diagnosis  will  be  found  on  p.  445. 


376  ANIMAL   PARASITES 

(2)  Treponema  pertenue,  morphologically  very  similar 
to  Treponema  pallidum,  was  found  by  Castellani  in 
yaws,  a  skin  disease  of  the  tropics. 

3.  Genus  Trypanosoma. — Trypanosomes  have  been 
found  in  the  blood-plasma  of  a  great  variety  of  verte- 
brates. Many  of  them  appear  to  produce  no  symptoms, 
but  a  few  are  of  great  pathologic  importance.  As  seen 
in  the  blood,  they  are  elongated,  spindle-shaped  bodies, 
the  average  length  of  dififerent  species  varying  from  10 
to  70  ;u.  Along  one  side  there  runs  a  delicate  undulating 
membrane,  the  free  edge  of  which  appears  to  be  some- 
what longer  than  the  attached  edge,  thus  throwing  it  into 
folds.  Somewhere  in  the  body,  usually  near  the  middle, 
is  a  comparatively  pale-staining  nucleus;  and  near  the 
posterior  end  is  a  smaller,  more  deeply  staining  chromatin 
mass,  the  micronucleus  or  blepharoplast.  A  number  of 
coarse,  deeply  staining  granules,  chromatophores,  may 
be  scattered  through  the  cytoplasm.  A  flagellum  arises 
in  the  blepharoplast,  passes  along  the  free  edge  of  the 
undulating  membrane,  and  is  continued  anteriorly  as 
a  free  flagellum.  These  details  of  structure  are  well 
shown  in  Plate  VII. 

The  life  history  of  the  trypanosomes  is  not  well  known. 
In  most  cases  there  is  an  alternation  of  hosts,  various 
insects  playing  the  part  of  definitive  host. 

Trypanosomes  have  been  much  studied  of  late,  and 
many  species  have  been  described.  At  least  three  have 
been  found  in  man. 

Trypanosoma  gamhiense  is  the  parasite  of  African 
"sleeping  sickness."  Its  detection  in  the  blood  is  de- 
scribed on  p.  295.  A  new  species  causing  sleeping  sick- 
ness in  man  has  recently  been  described  and  has  been 


PHYLUM   PROTOZOA  377 

named  T.  rhodesiense.  The  chief  point  of  distinction 
from  T.  gambiense  is  the  situation  of  the  nucleus  close 
to  or  even  posterior  to  the  blepharoplast.  It  is  trans- 
mitted by  the  fly  Glossina  morsitans. 

Trypanosoma  cruzi  is  a  small  form  which  has  been 
found  in  the  blood  of  man  in  Brazil. 


Fig.  114. — Trypanosoma  lewisi  in  blood  of  rat.  The  red  corpuscles  were  decolorized 
with  acetic  acid  (Xiooo)  (photograph  by  the  author  from  a  slide  presented  by  Prof. 
Novy). 

Trypanosoma  lewisi,  a  very  common  and  apparently 
harmless  parasite  of  gray  rats,  especially  sewer  rats,  is 
interesting  because  it  closely  resembles  the  pathogenic 
forms,  and  is  easily  obtained  for  study.  Its  posterior 
end  is  more  pointed  than  that  of  T.  gambiense  (Fig. 
114). 

Trypanosoma  evansi,  T.  brucei,  and  T.  eguiperdum 
produce  respectively  surra,  nagana,  and  dourine,  which 
are  common  and  important  diseases  of  horses,  mules, 
and  cattle  in  the  Philippines,  East  India,  and  Africa. 


378  ANIMAL   PARASITES 

4.  Genus  Leishmania.— The  several  species  which 
compose  this  genus  are  apparently  closely  related  to  the 
trypanosomes,  but  their  exact  classification  is  undeter- 
mined. They  have  been  grown  outside  the  body  and 
their  transformation  into  flagellated  trypanosome-Uke 
structures  has  been  demonstrated.  Calkins  places  them 
in  the  genus  Herpetomonas. 

(i)  Leishmania  donovani  is  the  cause  of  kala-azar,  an 
important  and  common  disease  of  India.  The  "Leish- 
man-Donovan  bodies"  are  round  or  oval  structures, 
2  to  3  f^  in  diameter,  with  two  distinct  chromatin  masses, 
one  large  and  pale,  the  other  small  and  deeply  staining. 
The  parasites  are  especially  abundant  in  the  spleen, 


Fig.  11$. — Cercomonas  hominis  (about   X500):  A,  Larger  variety;  B,  smaller  variety 

(Davaine). 

splenic  puncture  being  resorted  to  for  diagnosis.  They 
are  readily  found  in  smears  stained  by  any  of  the  Ro- 
manowsky  methods.  They  he  chiefly  within  endothe- 
lial cells  and  leukocytes.  They  are  also  present  within 
leukocytes  in  the  peripheral  blood,  but  are  difficult  to 
find  in  blood-smears. 

(2)  Leishmania  tropica  resembles  the  preceding.  It 
is  found,  lying  intracellularly,  in  the  granulation  tissue 
of  Delhi  boil  or  Oriental  sore. 

(3)  Leishmania  infantum  has  been  found  in  an  obscure 
form  of  infantile  splenomegaly  in  Algiers. 

5.  Genus  Cercomonas. — (i)  Cercomonas  hominis  has 
been  found  in  the  feces  in  a  variety  of  diarrheal  condi- 


PHYLUM   PROTOZOA 


379 


tions,  and  in  from  lo  to  25  per  cent,  of  healthy  persons  in 
tropical  regions.  It  is  probably  harmless.  The  body  is 
10  to  12  (.1  long,  is  pointed  posteriorly,  and  has  a  flagel- 
lum  at  the  anterior  end  (Fig.  115).  The  nucleus  is  diffi- 
cult to  make  out.  The  feces  should  be  examined  in  the 
fresh  state,  and  preferably  while  warm,  in  order  to  ob- 
serve the  active  motion  of  the  organism. 

6.  Genus  Bodo. — (i)  Bode  urinarius  is  sometimes  seen 
in  the  urine,  darting  about  in  various  directions.  It  is 
probably  an  accidental  contamination.     It  has  a  lancet- 


Fig.  116. — Trichomonas  vaginalis  (about  Xiooo)  (after  KoUikerand  Scanzoni). 


shaped  body,  about  10  u  long,  and  is  somewhat  twisted 
upon  itself,  with  two  fiagella  at  the  end. 

7.  Genus  Trichomonas. — (i)  Trichomonas  vaginalis. 
— The  acid  discharge  of  catarrhal  vaginitis  sometimes 
contains  this  parasite  in  abundance.  It  is  oval  or  pear 
shaped  and  has  a  cluster  of  fiagella  at  one  end  (Fig.  116). 
The  average  size  is  about  12  by  20  ;[*,  although  there  is 
considerable  variation  among  individuals.  As  seen  in 
fresh  material  it  may  suggest  an  actively  motile  pus- 
corpuscle.  When  in  motion  the  fiagella  are  not  easily 
seen.  No  pathogenic  significance  is  ascribed  to  it  in  the 
vagina,  but  a  few  cases  have  been  reported  in  which  it 


38o 


ANIMAL  PARASITES 


was  apparently  the  cause  of  a  urethritis  in  the  male.  It 
is  occasionally  found  in  the  urine.  This  and  similar  or- 
ganisms, such  as  cercomonas  and  bodo,  might  be  mis- 
taken for  spermatozoa  by  the  totally  inexperienced 
worker. 

(2)  Other  Trichomonads. — Various  forms  have  been 
described,  regarded  by  some  as  identical  with  T.  vagi- 


Fig.  117. — Lamblia  intestinalis  from  the  intestines  of  a  mou.-. 
and  Schweiakoff). 


i..bout  X  2000)  (Grassi 


nalis,  by  others  as  distinct  species.  Among  these  are 
T.  intestinalis,  sometimes  found  in  the  feces  in  diarrheal 
conditions,  and  T.  pulmonalis,  which  has  been  encoun- 
tered in  the  sputum  of  persons  suffering  from  pulmonary 
gangrene  and  putrid  bronchitis. 

8.  Genus  Lamblia. — (i)  Lamblia  intestinalis  is  a  very 
common  parasite  in  the  tropics,  but  is  generally  consid- 
ered of  little  pathogenic  importance.    It  is  pear  shaped, 


PHYLUM   PROTOZOA  38 1 

measures  lo  to  15  /t^  or  more  in  length,  and  has  a  depres- 
sion on  one  side  of  the  blunt  end,  by  which  it  attaches 
itself  to  the  tops  of  the  epithelial  cells  of  the  intestinal 
wall.  Three  pairs  of  flagella  are  arranged  about  the 
depression  and  one  pair  at  the  pointed  end  (Fig.  117). 
Unless  the  stool  is  obtained  by  catharsis  (see  p.  360), 
encysted  forms  only  may  be  found,  and  these  may  be 
difficult  or  impossible  to  recognize. 

SUBPHYLUM  SPOROZOA 
Class  Telosporidia 

All  the  members  of  this  class  are  parasitic,  but  only 
a  few  have  been  observed  in  man,  and  only  one  genus, 
Plasmodium,  is  of  much  importance  in  human  pathology. 
Propagation  is  by  means  of  spores,  and  sporulation  ends 
the  Hfe  of  the  individual.  In  some  species  there  is  an 
alternation  of  generations,  in  one  of  which  sexual  proc- 
esses appear.  In  such  cases  the  male  individual  may 
be  provided  with  flagella.  Otherwise,  there  are  no 
special  organs  of  locomotion. 

1.  Genus  Coccidium. — (i)  Coccidium  cuniculi. — This 
is  a  very  common  parasite  of  the  rabbit  and  has  been 
much  studied;  but  extremely  few  authentic  cases  of 
infection  in  man  have  been  reported.  The  parasite, 
which  when  fully  developed  is  ovoid  in  shape  and  meas- 
ures about  30  to  50  /W  in  length  and  has  a  shell-hke 
integument,  develops  within  the  epithelial  cells  of  the 
bile-passages.  Upon  reaching  adult  size  it  divides  into 
a  number  of  spores  or  merozoites  which  enter  other 
epithelial  cells  and  repeat  the  cycle.  A  sexual  cycle 
outside  the  body,  which  suggests  that  of  the  malarial 
parasite,  but  does  not  require  an  insect  host,  also  occurs. 


382  ANIMAL   PARASITES 

Infection   takes  place  from  ingestion  of  the  resulting 
sporozoites. 

2.  Genus  Plasmodium. — This  genus  includes  the  ma- 
larial parasites  which  have  already  been  described 
(see  p.  296). 

3.  Genus  Babesia. — The  proper  position  of  this  genus 
is  uncertain.  It  is  placed  among  the  flagellates  by  some. 
The  chief  member  is  Babesia  bigeminum,  the  cause  of 
Texas  fever  in  cattle.  It  is  a  minute,  pear-shaped  or- 
ganism, lying  in  pairs  within  the  red  blood-corpuscles. 
An  organism,  B.  (or  Piroplasma)  hominis,  described  as 
occurring  in  the  red  cells  in  "tick-fever"  of  Montana,  is 
also  placed  in  this  genus,  but  its  pathogenicity  and  even 
its  existence  are  questionable. 

SUBPHYLUM  INFUSORIA 
Class  Ciliata 

The  conspicuous  feature  of  this  class  is  the  presence 
of  cilia.  These  are  hair-Uke  appendages  which  have  a 
regular  to-and-fro  motion,  instead  of  the  irregular  lash- 
ing motion  of  flagella.  They  are  also  shorter  and  more 
numerous  than  flagella.  Most  infusoria  are  of  fixed 
shape  and  contain  two  nuclei.  Contractile  and  food- 
vacuoles  are  also  present.  Encystment  is  common. 
Only  one  species  is  of  medical  interest.  Certain  ciliated 
structures,  which  have  been  described  as  infusoria, 
notably  in  sputum  and  nasal  mucus,  were  probably 
ciUated  body  cells. 

I.  Genus  Balantidium. — (i)  Balantidium coli. — This 
parasite,  formerly  called  ParanuBcium  coli,  is  an  occa- 
sional inhabitant  of  the  colon  of  man,  where  it  some- 
times penetrates  into  the  mucous  membrane  and  pro- 


PHYLUM   PLATYHELMINTHES  383 

duces  a  diarrheal  condition  resembling  amebic  dys- 
entery. It  is  an  oval  organism,  about  60  to  100  ^i  long 
and  50  to  70  ^  wide,  is  covered  with  ciHa,  and  contains  a 
bean-shaped  macronucleus,  a  globular  micronucleus,  two 
contractile  vacuoles,  and  variously  sized  granules  (Fig. 
118). 


Fig.  118. — Balantidium  coli  (alx)ut  X  350)  (after  Eichhorst). 

Its  ordinary  habitat  is  the  rectum  of  the  domestic 
pig,  where  it  apparently  causes  no  disturbance.  It 
probably  reaches  man  in  the  encysted  condition. 

PHYLUM  PLATYHELMINTHES 

The  old  phylum  Vermidea  has  been  subdivided  into 
three  phyla,  those  which  are  of  interest  here  being 
the  Platyhelminthes  and  Nemathelminthes,  the  fiat 
worms  and  the  round  worms  respectively.  Of  these, 
many  species  are  parasitic  in  man  and  the  higher  ani- 
mals. In  some  cases  man  is  the  regular  host;  in  others 
the  usual  habitat  is  some  one  of  the  animals,  and  the 
occurrence  of  the  worm  in  man  is  more  or  less  acci- 
dental. Such  are  called  incidental  parasites.  Only  those 
worms  that  are  found  in  man  with  sufl&cient  frequency 
to  be  of  medical  interest  are  mentioned  here. 

The  most  important  means  of  clinical  diagnosis  of 
infection  by  either  the  flat  worms  or  the  round  worms 


384  ANIMAL   PARASITES 

is  the  finding  of  ova.  In  many  cases  the  ova  are  so 
characteristic  that  the  finding  of  a  single  one  will  estab- 
lish the  diagnosis.  In  other  cases  they  must  be 
carefully  studied  and  a  considerable  number  measured. 
While  ova  from  the  same  species  will  naturally  vary 
somewhat,  the  average  size  of  a  dozen  or  more  is  pretty 
constant.  The  measurements  given  here  are  mainly 
those  accepted  by  Stiles  or  Ward. 

PHYLUM   PLATYHELMINTHES 
(Flat  Worms) 
Class  Trematoda. — Flukes.    Unsegmented,  leaf  shaped. 

Genus.  Species. 

Fasciola.  F.  hepatica. 

Dicrocoelium.  D.  lanceatum. 

Opisthorchis.  Op.  felineus. 

Op.  sinensis. 
Fasciolopis.  F.  buski. 

Paragonimus.  P.  westermannii. 

Schistosomum.  S.  haematobium. 

S.  mansoni. 

S.  japonicum. 

Class  Cestoda. — Tapeworms.     Segmented,  ribbon  shaped. 
Genus.  Species. 

Taenia.  T.  saginata. 

T.  solium. 
T.  echinococcus. 
Hymenolepis.  H.  nana. 

H.  diminuta. 
Dipylidium.  D.  caninum. 

Dibothriocephalus.      D.  latus. 

Class  Trematoda 

The    trematodes,    commonly    known    as    "flukes," 

are  flat,  unsegmented,  generally  tongue-  or  leaf-shaped 

worms.     They  are  comparatively  small,  most  species 

averaging  between  5  and  15  mm.  in  length.    They  pos- 


PHYLUM  PLATYHELMINTHES  385 

sess  an  incomplete  digestive  tract,  without  anus,  and  are 
provided  with  one  or  more  sucking  disks  by  means  of 
which  they  can  attach  themselves  to  the  host.  Some 
are  also  provided  with  hooklets.  Nearly  all  species  are 
hermaphroditic,  and  the  eggs  of  nearly  all  are  opercu- 
lated  (provided  with  a  lid),  the  only  important  exception 
being  the  several  species  of  Schistosomum.  Development 
takes  place  by  alternation  of  generations,  the  interme- 
diate generation  occurring  in  some  water  animal:  mol- 


Fig.  119. — Fasciola  hepatica,  about  two-thirds  natural  size  (Mosler  and  Peiper). 

lusks,  amphibians,  fishes,  etc.     Trematode  infection  is 
uncommon  in  this  country. 

1.  Genus  Fasciola. — (i)  Fasciola  hepatica. — The 
"liver  fluke"  inhabits  the  bile-ducts  of  numerous  herbiv- 
orous animals,  especially  sheep,  where  it  is  an  important 
cause  of  disease.  It  brings  about  obstruction  of  the  bile- 
passages,  with  enlargement  and  degeneration  of  the  liver 
— "liver  rot."  A  species  of  snail  serves  as  intermediate 
host.  The  worm  is  leaf  shaped,  the  average  size  being 
about  2.8  by  1.2  cm.  The  anterior  end  projects  like  a 
beak  (head-cone  3  to  4  mm.  long)  (Fig.  119).  Ova  ap- 
pear in  the  feces.  They  are  yellowish  brown,  oval,  oper- 
culated,  and  measure  about  130  to  140  by  75  to  90  ^. 

25 


386  ANIMAL   PARASITES 

2.  Genus  Dicrocoelium. — (i)  Dicrocoelium  lanceatum 

is  often  associated  with  the  Hver  fluke  in  the  bile-passages 
of  animals,  but  is  neither  so  common  nor  so  widely 
distributed  geographically.  It  has  rarely  been  observed 
in  man.  It  is  smaller  (length  about  i  cm.)  and  more 
elongated.  The  eggs  measure  38  to  45  {^i  long  and  22  to 
30  fJ.  wide. 

3.  Genus  Opisthorchis. — (i)  Opisthorchis  felineus 
inhabits  the  gall-bladder  and  bile-ducts  of  the  domestic 
cat  and  a  few  other  animals.  Infection  in  man  has 
been  rcpeatedh^  observed  in  Europe,  and  especially  in 
Siberia.  The  body  is  flat,  yellowish-red  in  color,  and 
almost  transparent.  It  measures  8  to  11  by  1.5  to  2 
mm.  The  eggs,  which  are  found  in  the  feces,  are  oval, 
with  a  well-defined  operculum  at  the  narrower  end, 
and  contain  a  ciHated  embryo  when  deposited.  They 
measure  about  30  by  11  l-i. 

(2)  Opisthorchis  sinensis,  like  the  preceding  fluke, 
inhabits  the  gall-bladder  and  bile-ducts  of  domestic 
cats  and  dogs.  It  is,  however,  much  more  frequent  in 
man,  being  a  conmion  and  important  parasite  in  certain 
parts  of  Japan  and  China.  The  number  present  may 
be  very  great;  over  4000  were  counted  in  one  case. 
The  parasite  resembles  Op.  felineus  in  shape  and  color. 
It  is  10  to  14  mm.  long  and  2.5  to  4  mm.  broad.  The 
eggs  have  a  sharply  defined  Hd  and  measure  25  to  30  by 
15  to  17  ,M.  When  they  appear  in  the  feces  they  con- 
tain a  ciliated  embr>'o.  The  intermediate  host  is  un- 
known. 

4.  Genus  Fasciolopis. — (i)  Fasciolopis  buski. — 
This  fluke  is  parasitic  in  the  duodenum  of  man,  and  is 
widespread  in  the  East,  notably  in  India,  China,  and 


PHYLUM   PLATYHELMINTHES  387 

Japan.  A  few  imported  cases  have  been  reported  in 
this  country.  When  in  considerable  numbers  it  causes 
a  bloody  diarrhea  accompanied  by  high  fever.  The 
usual  length  is  about  30  mm.;  width,  10  to  12  mm.; 
thickness,  1.5  to  4  mm.  The  eggs  are  thin  shelled,  with 
granular  contents,  possess  a  minute  operculum,  and 
measure  about  125  by  75  to  So  u. 

5.  Genus  Paragonimus.— (i)  Paragonimus  wester- 
mannii,  called  the  "lung  fluke,"  is  also  a  common  parasite 
of  man  in  Japan,  China,  and  Korea.  It  inhabits  the 
lung,  causing  the  formation  of  small  cavities.  Mod- 
erate hemoptysis  is  the  principal  symptom.  Ova  are 
readily  found  in  the  sputum  (Fig.  120) ;  the  worms  them- 
selves are  seldom  seen,  except  postmortem.  The  worms 
are  faint  reddish-brown  in  color,  egg  shaped,  with  the 
ventral  surface  flattened,  and  measure  8  to  10  by  4  to 
6  mm.  The  ova  are  thin  shelled,  operculated,  brownish 
yellow,  and  measure  about  87  to  100  by  52  to  66  ii. 
Little  is  known  of  the  development  outside  the  body. 

According  to  Ward,  three  distinct  species  have  been 
confused  under  the  name  P.  westermannii:  the  original 
form,  P.  westermannii,  found  in  the  tiger;  the  American 
lung  fluke,  P.  kellicotti,  thus  far  found  only  in  cat,  dog, 
and  hog;  and  the  Asiatic  lung  fluke  of  man,  P.  ringeri, 
described  above. 

6.  Genus  Schistosomum. — (i)  Schistosomum  haema- 
tobium.— This  trematode,  frequently  called  Bilharzia 
hcBmatobia,  is  an  extremely  common  cause  of  disease 
(bilharziasis  or  Egyptian  hematuria)  in  northern  Africa, 
particularly  in  Egypt. 

Unlike  the  other  flukes,  the  sexes  are  separate.  The 
male  is  12  to  14  mm.  long  and  i  mm.  broad.    The  body 


388 


ANIMAL  PARASITES 


is  flattened  and  the  lateral  edges  curl  ventrally,  forming  a 
longitudinal  groove,  in  which  the  female  lies  (Fig.  122). 
The  latter  is  cyhndric  in  shape,  about  20  mm.  long  and 
0.25  mm.  in  diameter.  The  eggs  are  an  elongated  oval, 
about  120  to  190  fi  long  and  50  to  73  fJ-  broad,  yellowish 
in  color,  and  slightly  transparent.  They  possess  no  hd, 
such  as  characterizes  the  eggs  of  most  of  the  trematodes, 


Fig.  120. — Sputum  of  man  containing  eggs  of  the  lung  fluke,  greatly  enlarged.     (After 

Manson.) 

but  are  provided  with  a  thorn-Uke  spine  which  is  placed 
at  one  end  (Fig.  121).     Within  is  a  ciliated  embryo. 

In  man  the  worm  lives  in  the  veins,  particularly  the 
portal  vein  and  the  veins  of  the  bladder  and  rectum, 
leading  to  obstruction  and  inflammation.  The  eggs 
penetrate  into  the  tissues  and  are  present  in  abundance 
in  the  mucosa  of  the  bladder  and  rectum.  They  also 
appear  in  the  urine  and,  less  commonly,  in  the  feces. 
The  mode  of  infection  is  unknown. 


PHYLUM  PLATYHELMINTHES 


389 


(2)  Schistosomum  mansoni. — It  has  long  been  ob- 
served that  schistosomum  eggs  in  the  urine  have  usually 
a  terminal  spine,  while  in  the  feces 
the  lateral  spine  is  more  common. 
It  is  now  known  that  the  lateral- 
spined  egg  is  that  of  a  distinct 
species,  to  which  the  name  Schisto- 
somum mansoni  has  been  given. 
It  is  found  in  Africa  along  with 
Schistosomum  hcsmatobium,  but  is 
especially  prevalent  in  the  West 
Indies  and  Central  America.  The 
adult  worms  closely  resemble  the 
male  and  female  of  S.  hcematobium. 
They  inhabit  the  rectal  and  portal 
veins,  and  ova  appear  in  the  feces, 
where  they  are  very  easily  recog- 
nized from  their  size  and  the  char- 
acteristic spine  (see  Figs.  123  and 
135).  They  are  light  yellow  in 
color,  measure  112  to  162  by  60  to  70  ^,  and  are  pro- 
vided with  a  cleanly  cut,  sharply  pointed  spine,  which 
is  situated  at  the  juncture  of  the  last  and  third  quarter 


Fig.  121. — Egg  of  human 
blood  fluke  {Schistosomum 
hamalobium),  with  con- 
tained embryo,  passed  in 
the  urine  (X  285).  (After 
Looss.) 


Fig.  122.— Schistosomum  haematobium,  male  and  female  (about  X  4).  with  egg  (about 
X  70)  (von  Jaksch). 

of  the  egg  and  is  directed  backward.  Within  the  egg 
is  a  ciliated  embryo  (miracidium)  which  can  be  seen 
without  difficulty. 


390 


ANIMAL   PARASITES 


(3)  Schistosomum  japonicum  resembles  5.  hamato- 
hium  morphologically,  but  both  the  male  and  female  are 
smaller.    The  ova,  which  appear  in  the  feces,  are  ovoid, 


Fig.  123. — Ova  of  Schistosomum  mansoni:  i,  With  spine  out  of  focus;  2,  in  a  clump 
of  red  blood-cells;  3,  apparently  unfertilized;  4,  usual  appearance  (X  250).  (Photo- 
graphs by  the  author.) 

thin  shelled,  and  without  lid  or  spine.  They  average 
^2>  by  62  {-I  in  size,  and  contain  a  ciliated  embryo.  The 
worm  inhabits  the  portal  and  probably  also  other  veins. 

Class  Cestoda 

The  cestodes,  or  tapeworms,  are  very  common  para- 
sites of  both  man  and  the  animals.    In  the  adult  stage 


PHYLUM   PLATYHELMINTHES  39I 

they  consist  of  a  linear  series  of  flat,  rectangular  seg- 
ments (proglottides) ,  at  one  end  of  which  is  a  smaller 
segment,  the  scolex  or  head,  especially  adapted  by  means 
of  sucking  disks  and  hooklets  for  attachment  to  the  host. 
The  series  represents  a  colony,  of  which  the  scolex  is 
ancestor.  The  proglottides  are  sexually  complete  in- 
dividuals (in  most  cases  hermaphroditic)  which  are 
derived  from  the  scolex  by  budding.  With  the  excep- 
tion of  the  immature  segments  near  the  scolex,  each 
contains  a  uterus  filled  with  ova. 

The  large  tapeworms,  Tcenia  saginata,  T.  solium,  and 
Dihothriocephalus  latus,  are  distinguished  from  one  an- 
other mainly  by  the  structure  of  the  scolex  and  the 
uterus.  The  scolex  should  be  studied  with  a  low- 
power  objective  or  a  hand  lens.  The  uterus  is  best 
seen  by  pressing  the  segment  out  between  two  plates 
of  glass. 

All  the  tapeworms  pass  a  larval  stage  in  the  tissues  of 
an  intermediate  host,  which  is  rarely  of  the  same  species 
as  that  which  harbors  the  adult  worm.  From  the  ova 
which  have  developed  in  the  proglottides  of  the  adult 
worm,  and  which  pass  out  with  the  feces  of  the  host, 
there  develop  embryos,  or  oncospheres,  each  provided 
with  three  pairs  of  horny  hooklets.  When  the  oncosphere 
is  taken  into  the  intestines  of  a  suitable  animal,  it  pene- 
trates to  the  muscles  or  viscera  and  there  forms  a  cyst 
in  which  develop  usually  one,  but  sometimes  many, 
scoUces,  which  are  identical  with  the  head  of  the  adult 
worm.  When  the  flesh  containing  this  cystic  stage  is 
eaten  without  sufficient  cooking  to  destroy  the  scolices, 
the  latter  attach  themselves  to  the  intestinal  wall  and 
produce  adult  tapeworms  by  budding. 


392 


ANIMAL   PARASITES 


Ordinarily,  only  the  adult  stage  occurs  in  man.  In  the 
case  of  TcBtiia  echinococcus  only  the  larval  stage  is  found. 
T.  solium  may  infect  man  in  either  stage,  although  the 
cystic  stage  is  rare. 

Since  the  head,  or  scolex,  is  the  ancestor  from  which 
the  worm  is  formed  in  the  intestine,  it  is  important, 
after  giving  a  vermifuge,  to  make  certain  that  the  head 
has  been  passed  with  the  worm.  Should  it  remain,  a 
new  worm  will  develop. 

The  principal  tapeworms  found  in  man  belong  to 
the  genera  Taenia,  Hymenolepis,  and  Dibothriocephalus. 


v^cfl.iiiinnHfjjjjiiiiiiruMiiiimui 
%IIlllllllll||||p^^^ 


Q] 


•Lm.:i:rrj 

Fig.  124. — Taenia  saginata  (Eichhorst). 


1.  Genus  Taenia. — (i)  Taenia  saginata  (Fig.  124). — 
This,  the  beef  tapeworm,  is  the  common  tapeworm  of  the 
United  States,  and  is  widely  distributed  over  the  world. 
Its  length  is  generally  about  4  to  8  meters.  The  scolex 
is  about  the  size  of  a  large  pin-head  (1.5  to  2  mm.  in 
diameter) ,  and  is  surrounded  by  four  sucking  disks,  but 
has  no  booklets  (Fig.  125).  The  neck  is  about  i  mm. 
wide.  The  terminal  segments,  which  become  detached 
and  appear  in  the  feces,  measure  about  18  to  20  mm. 
long  by  4  to  7  mm.  wide.    The  uterus  extends  along  the 


PHYLXJM   PLATYHELMINTHES  393 

midline  of  the  segment  and  gives  off  twenty  to  thirty 
branches  upon  each  side  (see  Fig.  133,  i). 

The  larval  stage  is  passed  in  the  muscles  of  various 
animals,  especially  cattle.  It  rarely  or  never  occurs  in 
man,  hence  there  is  little  or  no  danger  of  infection  from 
examining  feces. 

The  scolex  is  ingested  with  the  meat,  its  capsule  is 
dissolved  by  the  digestive  juices,  and  it  attaches  itself  to 
the  intestinal  wall  by  means  of  its  suckers.     It  then 


Fig.  125. — Head  of  Ta:nia  saginata  (Mosler  and  Peiper). 

develops  into  the  mature  worm,  which  may  grow  very 
rapidly,  even  as  many  as  thirteen  or  fourteen  segments 
being  formed  in  a  day. 

The  ova  are  present  in  the  feces  of  infected  persons, 
sometimes  in  great  numbers.  When,  however,  segments 
are  passed,  the  ova  for  the  most  part  remain  within 
them  and  comparatively  few  are  found  free  in  the  feces. 
They  are  spheric  or  ovoid,  yellow  to  brown  in  color, 
and  have  a  thick,  radially  striated  shell  (Fig.  126). 
Within  them  the  six  booklets  of  the  embryo  (oncosphere) 
can  usually  be  made  out  as  three  pairs  of  parallel  lines. 


394 


ANIMAL   PARASITES 


The  size  of  the  ova  varies  from  20  to  30  (J.  wide  and 
30  to  40  ^  long.     Vegetable  cells,  which  are  generally 


Kig.  126. — EgRs  of  Taenia  saginata,  magnifications  loo,  250,  and  500  diameters  (photo- 
graphs by  the  author). 

present    in    the  feces,  are    often   mistaken  for  them, 
although  there  is  no  great  resemblance. 


Fig.  127. — Head  of  Taenia  solium  (Mosler  and  Peiper).  4 

(2)  Taenia  solium,  the  pork  tapeworm,  is  very  rare 
in  this  country.  It  is  usually  much  shorter  than  Tania 
saginata.     The  scolex  is  about  0.6  to  i  mm.  wide,  is 


PHYLUM   PLATYHELMINTHES 


395 


surrounded  by  four  sucking  disks,  and  has  a  projection, 
or  rostellum,  with  a  double  row  of  horny  booklets,  usually 
twenty-six  to  twenty-eight  in  number.  (Fig.  127).  The 
terminal  segments  measure  about  5  to  6  by  10  to  12 
mm.  The  uterus  has  only  seven  to  fourteen  branches 
on  each  side  (see  Fig.  133,  3). 

The  cysticercus  stage  occurs  ordinarily  in  the  muscles 
of  the  pig,  but  is  occasionally  seen  in  man,  most  fre- 
quently affecting  the  brain  and  eye  {Cysticercus  cellu- 
Ioscb).  There  is,  therefore,  danger  of  infection  from 
handhng  feces. 

The  ova  so  closely  resemble  those 
of  Tcenia  saginata  as  to  be  prac- 
tically indistinguishable.  They  aver- 
age about  31  to  36  M  in  diameter  and 
are  usually  spheric. 

(3)  Taenia  echinococcus.  —  The 
mature  form  of  this  tapeworm  in- 
habits the  intestines  of  the  dog  and 
wolf.  The  larvae  develop  in  cattle 
and  sheep  ordinarily,  but  are  some- 
times found  in  man,  where  they  give 
rise  to  echinococcus  or  "hydatid" 
disease.  The  condition  is  unusual  in  America,  but  is 
not  infrequent  in  Central  Europe  and  is  common  in 
Iceland  and  Australia. 

The  adult  parasite  is  2.5  to  5  mm.  long  and  consists 
of  only  four  segments  (Fig.  128).  It  contains  many  ova. 
When  the  ova  reach  the  digestive  tract  of  man  the  em- 
bryos are  set  free  and  find  their  way  to  the  liver,  limg, 
or  other  organ,  where  they  develop  into  cysts,  thus 
losing  their  identity.    The  cysts  may  attain  the  size  of 


Fig.  128. — Tania  echi- 
nococcus; enlarged  (Mos- 
ler  and  Peiper). 


396 


ANIMAL  PARASITES 


a  child's  head.  Other  cysts,  called  "daughter-cysts," 
are  formed  within  these.  The  cyst-wall  is  made  up  of 
two  layers,  from  the  inner  of  which  develop  larvae  which 
are  identical  with  the  head,  or  scolex,  of  the  mature 
parasite.  These  are  ovoid  structures  0.2  to  0.3  mm. 
long.     Each  has  four  lateral  suckers  and  a  rostellum 


!^%;J:;''x*V;'<  V"''. 


k^> 


i^'vl'?^ 


^ 

^ 


Fig.  129. — Scolex  and  booklets  of  Tania  cxhinococcus  iu  fluid  from  hepatic  cyst  (X300) 
(photographs  by  the  author). 

surmounted  by  a  double  circular  row  of  horny  hooklets. 
The  rostellum  with  its  hooklets  is  frequently  invaginated 
into  the  body. 

Diagnosis  of  echinococcus  disease  depends  upon  de- 
tection of  scolices,  free  hooklets  which  have  fallen  off 
from  degenerated  scolices,  or  particles  of  cyst-wall  which 


PHYLUM   PLATYHELMINTHES  397 

are  characteristically  laminated  and  usually  have  curled 
edges.  The  lamination  is  best  seen  at  the  torn  edge  of 
the  membrane.  All  of  these  structures  can  be  found  in 
fluid  withdrawn  from  the  cysts  or,  less  frequently,  in 
the  sputum  or  the  urine,  when  the  disease  involves  the 
lung  or  kidney  (see  Figs.  59  and  129).  The  cysts  are 
sometimes  "barren,"  growing  to  a  considerable  size 
without  producing  scolices. 

The  cyst  fluid  is  clear,  between  1.009  ^.nd  1.015  in 
specific  gravity,  and  contains  a  notable  amount  of  so- 
dium chlorid,  but  no  albumin. 

2.  Genus  Hymenolepis. — (i)  Hymenolepis  nana,  the 
dwarf  tapeworm  (Fig.  130),  is  i  to  4.5  cm.  in  length 


Fig.  130. — Hymenolepis  nana,  about  Fig.    131. — Egg    of    hymenolepis    nana, 

natural  size  (Hosier  and  Peiper).  as  seen  in  fresh  feces.     Enlarged.     (After 

Ransom,  from  Stiles.) 

and  0.5  to  0.7  mm.  in  breadth  at  the  widest  part.  The 
head  is  globular  and  has  a  rostellum  with  a  crown  of  24 
to  30  booklets.  There  are  about  150  segments.  Diag- 
nosis must,  in  general,  depend  upon  the  discovery  of  ova 
in  the  feces.  These  are  spheric  and  have  two  distinct 
membranous  walls.  The  outer  membrane  is  about  39  ^ 
in  diameter.  The  inner  averages  28  fi,  and  at  each  pole 
has  a  slight  projection  provided  with  indistinct  fila- 
mentous processes,  which  may  lie   between   the   two 


398  ANIMAL   PARASITES 

membranes  in  such  a  way  as  to  simulate  a  third.  The 
egg  contains  an  embryo,  of  which  only  the  three  pairs 
of  booklets  are  clearly  seen. 

The  worm  is  common  in  Europe  and  America.  It 
is  most  frequent  in  children  and  is  generally  present  in 
large  numbers,  producing  considerable  digestive  and 
nervous  disturbances.  The  mode  of  infection  is  un- 
known. 

(2)  Hjnnenolepis  diminuta  is  a  common  intestinal 
parasite  of  rats.  A  few  cases  of  infection  in  man  have 
been  reported  in  America.  The  parasite  measures  i  to 
6  cm.  in  length  and  is  composed  of  600  to  1300  segments. 
The  scolex  lacks  booklets.  Ova  are  round  or  ovoid,  with 
a  distinct,  sometimes  radially  striated  outer  membrane, 
56  to  80  i^i  in  diameter,  and  an  inner  membrane  24  to 
40  by  20  to  35  i^i.  Between  these  membranes  is  a  broad 
layer  of  gelatinous  substance.  The  contained  embryo 
has  six  booklets. 

3.  Genus  Dipylidium. — (i)  Dipylidium  caninum, 
sometimes  called  Tania  elliptica,  is  a  very  common  tape- 
worm of  dogs  and  cats.  Its  length  is  15  to  35  cm.  The 
head,  globular  in  shape,  is  armed  with  booklets.  Ter- 
minal segments  are  shaped  like  melon  seeds,  8  to  11  mm. 
long  and  1.5  to  3  mm.  broad.  Ova  are  spheric,  43  to 
50  u  in  diameter,  and  thin  shelled.  They  contain  a  six- 
hooked  embryo,  32  to  36  u  in  diameter. 

The  intermediate  host  is  the  flea  or  louse.  Infection 
of  human  beings  is  not  common,  and  is  mostly  confined 
to  children,  who  are  probably  infected  from  getting  lice 
or  fleas  of  dogs  or  cats  into  their  mouths. 

4.  Genus  Dibothriocephalus.— (i)  Dibothrioceph- 
alus   latus,   the  fish  tapeworm,  sometimes  reaches   20 


PHYLUM   PLATYHELMINTHES  399 

meters  in  length,  although  it  is  generally  not  more 
than  one-half  or  one-third  as  long.  When  several 
worms  are  present,  they  are  much  shorter,  often  only 


Fig.  132. — Head  of  Dibothriocephalus  latus  (about  X  9):  a,  a,  Head  grooves;  b,  neck 

(Blanchard). 

1.5  or  2  meters.  The  head  is  oval,  about  i  mm.  broad 
and  1.5  mm.  long.  It  is  unprovided  with  either  suckers 
or  hooklets,  but  has  two  longitudinal  grooves  which 


I  2  3 

Fig.  133.— Segments  of— (i)  Ta;nia  saginata;  (2)  Dibothriocephalus  latus;   {3)  Taenia 
solium,  showing  arrangement  of  uterus. 

serve  the  same  purpose  (Fig.  132).  The  length  of  the 
segments  is  generally  less  than  their  breadth,  mature 
segments  measuring  about  3  by  10  or  12  mm.     The 


400  ANIMAL  PARASITES 

uterus,  which  is  situated  in  the  center  of  the  segment, 
is  roset  shaped  (Fig.  133,  2)  and  brown  or  black  in  color. 

The  number  of  segments  sometimes  exceeds  3000. 
As  a  rule  they  do  not  appear  in  the  feces  singly,  but  in 
chains  of  considerable  length. 

The  larval  stage  is  found  in  various  fish,  notably  the 
pike,  burbot,  grayling,  and  certain  trout.  Infection  of 
man  prevails  only  in  regions  where  these  fish  are  found. 


• 


Fig.  134. — Ova  of  Dibothriocephalus  latus  (X  2$o  and  500).    The  lids  were  forced  open 
by  pressure  upon  the  cover-glass  (photographs  by  the  author). 

It  is  very  common  in  Japan  and  in  various  countries  of 
Europe,  especially  Ireland  and  the  Baltic  provinces  of 
Russia.  A  number  of  cases  of  infection  have  been  re- 
ported in  this  country,  a  few  of  which  were  undoubtedly 
acquired  here.  Any  locality  in  which  favorable  fish  are 
native  becomes  a  possible  center  of  infection  if  the  worm 
is  introduced  by  infected  immigrants. 

The  ova  are  characteristic.  They  measure  about  45 
by  70  //,  are  brown  in  color,  and  are  filled  with  small 
spherules.  The  shell  is  thin  and  has  a  small  hinged  lid 
at  one  end.    As  the  eggs  appear  in  the  feces  the  Hd  is 


Fig- 135-— Showing  comparative  size  of  ova  found  in  the  feces:  i,  Trichocephalus  tri- 
chiurus;  2,  Ascaris  lumbricoides;  3.  Necator  americanus,  four-cell  stage;  4,  Schistoso- 
mum  mansoni;  5,  Taenia  saginata;  6,  Dibothriocephalus  lat^,^tj^e  ^ipeof  ,tl>e  li4  Ijeing 
out  of  focus  ( X  250).     (Photographs  by  the  author.)  -,,-,,  1  -, 


402  ANIMAL   PARASITES 

not  easily  seen,  but  it  may  be  demonstrated  by  sufficient 
pressure  upon  the  cover-glass  to  force  it  open  (Figs.  134, 
135).  The  only  other  operculated  eggs  met  with  in 
man  are  those  of  the  fluke  worms. 

Diholhriocephalus  latus  is  interesting  clinically  because 
it  often  causes  a  very  severe  grade  of  anemia,  which  may 
be  indistinguishable  from  pernicious  anemia. 

PHYLUM  NEMATHELMINTHES 


(Round  Worms) 

atoda. — Unsegmented,  cylindric  or  fusif 

Genus. 

Species. 

Anguillula. 

A.  aceti. 

Ascaris. 

A.  lumbricoides. 

A.  canis. 

Oxyuris. 

0.  vermicularis. 

Filaria. 

F.  bancrofti. 

F.  philippinensis. 

F.  perstans. 

F.  diuma. 

F.  medinensis. 

Uncinaria. 

U.  duodenalis. 

Necator. 

N.  americanus. 

Strongyloides. 

S.  intestinalis. 

Trichinella. 

T.  spiralis. 

Trichocephalus. 

T.  trichiurus. 

Class  Nematoda 

The  nematodes,  or  round-worms,  are  cylindric  or  fusi- 
form worms,  varying  in  length,  according  to  species, 
from  I  mm.  to  40  or  80  cm.  As  a  rule,  the  sexes  are 
separate.  The  male  is  smaller  and  more  slender  than 
the  female.  In  a  few  cases  the  female  is  viviparous;  in 
most  cases  she  deposits  ova  which  are  characteristic, 
so  that  the  finding  of  a  single  egg  may  establish  the 
diagnosis.     Except  in  a  few  instances  the  young  are 


PHYLUM   NEMATHELMINTHES 


403 


different  from  the  adult,  and  must  pass  a  certain  larval 
stage  of  development  before  again  reaching  a  host. 
An  intermediate  host  is,  however,  necessary  with  only 
a  few  species. 

1.  Genus  Anguillula. 
— (i)  Anguillula  aceti. — 
This  worm,  commonly 
called  the  "vinegar  eel," 
is  usually  present  in  vine- 
gar. A  drop  of  the  vine- 
gar, particularly  of  the 
sediment,  will  frequently 
show  great  numbers,  all 
in  active  motion:  males, 
about  I  or  1.5  mm.  long; 
females,  somewhat  larger 
and  frequently  containing 
several  coiled  embryos; 
and  young,  of  all  sizes  up 
to  the  adult  (see  Fig.  60). 

The  vinegar  eel  is  never 
parasitic,  but  is  occasion- 
ally met  with  as  a  con- 
tamination  in    the    urine 

(see  p.  208),  and  has  there  been  mistaken  for  the  larva 
of  filaria  or  strongyloides. 

2.  Genus  Ascaris. — (i)  Ascaris  lumbricoides. — The 
female  is  20  to  40  cm.  long  and  about  5  mm.  thick  (Fig. 
136);  the  male,  15  to  17  cm.  long  and  3  mm.  thick. 
They  taper  to  a  blunt  point  anteriorly  and  posteriorly. 
At  the  anterior  end  are  three  small  lips  which  can  easily 
be  seen  with  a  hand  lens.    Their  color  is  reddish  or  brown. 


Fig.  136. — Ascaris  lumbricoides  (female) 
(Hosier  and  Peiper). 


404  ANIMAL  PARASITES 

They  are  the  common  "round-worms"  so  frequently 
found  in  children.  Their  habitat  is  the  small  intestine. 
Usually  several  individuals  are  present  and  sometimes 
many. 

The  diagnosis  is  made  by  detection  of  the  worms  or  ova 
in  the  feces.  The  latter  are  generally  numerous  and  are 
easily  recognized.  They  are  elliptic,  measuring  about 
49  by  65  ^,  and  have  an  unsegmented  protoplasm. 
There  is  usually  a  crescentic  clear  space  at  each  pole 
between  the  contents  and  the  shell  (Fig.   137).     The 


i 


# 


m 


Fig.  137. — Ova  of  Ascaris  lumbricoides  in  fresh  feces  (X  23°)  (photographs  by  the 

author). 

shell  is  thick  and  has  a  roughly  mammillated  or  sculp- 
tured surface  (Fig.  138).  When  only  females  are  pres- 
ent in  the  intestine,  and  occasionally  at  other  times, 
one  finds  unfertilized  eggs.  These  are  generally  much 
more  elongated,  have  a  thinner  and  smoother  shell, 
have  coarsely  granular  contents,  and  lack  the  cres- 
centic clear  spaces. 

The  eggs  do  not  hatch  in  the  intestine  of  the  original 
host.  They  pass  out  in  the  feces  and,  after  a  variable 
period,  usually  about  five  weeks,  come  tc  contain  an 


PHYLUM   NEMATHELMINTHES 


405 


embryo  which  remains  within  the  shell  until  ingested 
by  a  new  host.  The  embryo  is  very  resistant  and  may 
remain  alive  within  the  shell  for  years  even,  according 
to  Morris,  when  preserved  in  2  per  cent,  formalin. 
Upon  reaching  the  intestine  of  the  new  host  it  hatches 
out  and  develops  into  the  adult  worm  in  about  a 
month. 

(2)  Ascaris  canis  is  the  very  common  "stomach  worm" 
of  cats  and  dogs.  It  is  also  known  as  Ascaris  mystax 
and  Toxocara  canis.     It  is  rare  as  a  human  parasite. 


Fig.  138. — Egg  of  Ascaris   lumbricoides,  surface  view  (X  250)   (photograph  by  the 

author). 

The  male  is  4  to  9  cm.  long;  the  female,  12  to  20.  Indi- 
viduals from  dogs  are  generally  larger  than  those  from 
cats.  The  egg  is  spheric,  68  to  70  fJ.  in  diameter,  and 
has  a  thin  shell  with  comparatively  smooth  surface. 

3.  Genus  Oxyuris. — (i)  Oxyuris  vermicularis.— This 
is  the  "thread-worm"  or  "pin-worm"  which  in  the  adult 
stage  inhabits  the  colon  and  rectum,  especially  of  young 
children.  Its  presence  should  be  suspected  in  all  unex- 
plained cases  of  pruritus  ani.  The  female  is  about  9  to 
12  mm.  long;  the  male,  about  3  to  5  mm.  (Fig.  139). 

The  worms  are  not  infrequently  found  in  the  feces; 


406  ANIMAL   PARASITES 

the  ova,  rarely.  The  latter  are  best  found  by  scraping  the 
skin  with  a  dull  knife  at  the  margin  of  the  anus,  where 
they  are  deposited  by  the  female,  who  wanders  out  from 
the  rectum  for  this  purpose,  thus  producing  the  trouble- 
some itching.  They  are  asymmetrically  oval  with  one 
flattened  side,  are  about  50  (J.  long  by  16  to  20  /u  wide, 
have  a  thin  clear  shell,  and  when  desposited  contain  a 
partially  developed  embryo.  The  diagnosis  is  best  made 
by  giving  a  purgative  and  searching  the  stool  for  the 
adult  worms. 


Fig.  139. — Oxyuris  vermicularis,  male  and  female,  natural  size  (after  HeUer). 

Infection  takes  place  through  swallowing  the  ova. 
Auto-infection  is  likely  to  occur  in  children;  the  ova 
cling  to  the  fingers  after  scratching  and  are  thus  carried 
to  the  mouth.  Diagnosis  can  sometimes  be  made  by 
finding  the  ova  in  the  dirt  beneath  the  finger-nails. 

4.  Genus  Filaria. — (i)  Filaria  bancrofti. — The  adults 
are  thread-like  worms,  the  male  about  4  cm.,  the  female 
about  8  cm.,  long.  They  live  in  pairs  in  the  lymphatic 
channels  and  glands,  especially  those  of  the  pelvis  and 
groin,  and  often  occur  in  such  numbers  as  to  obstruct 
the  flow  of  lymph.  This  is  the  most  common  cause  of 
elephantiasis.  Infection  is  very  common  in  tropical 
countries,  especially  in  Samoa,  the  West  Indies,  Central 
America,  and  the  Isthmus  of  Panama.  It  is  said  that  in 
Samoa  50  per  cent,  of  the  natives  are  infected. 


PHYLUM   NEMATHELMINTHES  407 

The  female  is  viviparous,  and  produces  vast  numbers 
of  larvae,  which  appear  in  the  circulating  blood.  The 
name  Filaria  sanguinis  hominis,  which  is  commonly 
applied  to  them,  is  incorrect,  since  they  do  not  consti- 
tute a  species.  These  larvae  are  about  as  wide  as  a  red 
corpuscle  and  0.2  to  0.4  mm,  long  (see  Fig.  103),  and  are 
very  active,  although,  owing  to  the  fact  that  they  are 
inclosed  in  a  loose  transparent  sheath,  they  do  not  move 


Fig.  140. — Larva  of  Filaria  bancrofti  in  chylous  hydrocele  fluid;  length,  300  j*;  width, 
8  M-  A  number  of  red  blood-corpuscles  also  appear  (studied  through  courtesy  of  Dr. 
S.  D.  Van  Meter). 

about  from  place  to  place.  They  are  found  in  the  pe- 
ripheral blood  only  at  night,  appearing  about  8  p.  m., 
and  reaching  their  maximum  number — which  is  some- 
times enormous — about  midnight.  If  the  patient  change 
his  time  of  sleeping,  they  will  appear  during  the  day. 
Infection  is  carried  by  a  variety  of  mosquito,  which 
acts  as  intermediate  host.  Diagnosis  rests  upon  detec- 
tion of  larvae  in  the  blood,  as  described  on  p.  304. 

The  larvae  are  sometimes  found  in  urine  and  chylous 
fluids  from  the  serous  cavities.     Their  motion  is  then 


4o8  ANIMAL   PARASITES 

usuaUy  less  active  than  when  m  blood.  That  shown 
in  Fig.  140  was  alive  sixty  hours  after  removal  of  the 
fluid.  Larvae  were  present  in  the  blood  of  the  same 
patient. 

A  number  of  other  filariae  whose  larvae  appear  in  the 
blood  are  known,  some  of  them  only  in  the  larval  stage. 
Among  these  are  Filaria  philippinensis  and  F.  perstans, 
which  exhibit  no  periodicity,  and  F.  diurna  and  F.  ha, 
whose  larvae  appear  in  the  blood  during  the  day.  The 
adult  of  the  last  named  is  especially  frequent  in  the 
orbit  and  beneath  the  conjunctiva. 

(2)  Filaria  medinensis,  the  "guinea-worm,"  is  a  very 
interesting  and  important  worm  of  Africa  and  southern 
Asia.  It  has  been  thought  to  be  the  "fiery  serpent" 
which  molested  the  Children  of  Israel  in  the  Wilderness. 

The  larva  probably  enters  the  body  through  the  skin 
or  gastro-intestinal  tract.  It  wanders  about  in  the  sub- 
cutaneous tissues  until  maturity,  producing  sUght,  if  any, 
symptoms.  The  male  has  only  recently  been  discovered. 
It  is  only  4  cm.  long.  It  dies  soon  after  the  female  is 
impregnated.  The  adult  female  is  a  very  slender, 
yellowish  worm,  about  50  to  80  cm.  long,  its  appearance 
somewhat  suggesting  a  catgut  suture.  When  gestation 
is  complete  the  greater  part  of  the  female's  body  consists 
of  a  uterus  filled  with  embryos.  The  female  then  travels 
to  the  feet  or  ankles  of  the  host  and  there  produces  a  red 
nodule  and,  finally,  an  ulcer,  from  the  center  of  which 
her  head  protrudes.  Through  this  great  numbers  of 
larvae  are  discharged  whenever  it  comes  in  contact 
with  water.  Little  damage  is  done  unless  the  worm  is 
pulled  out,  when  the  larvae  are  set  free  in  the  tissues 
and  cause  serious  disturbances. 


PHYLUM  NEMATHELMINTHES 


409 


When  discharged  the  larvae  seek  out  a  small  crus- 
tacean, Cyclops,  which  serves  as  intermediate  host. 
5.  Uncinaria  duodenalis  and  Necator  americanus. 

— These,  the  Old  and  the  New  World  hookworm,  re- 
spectively, are  among  the  more  harmful  of  the  animal 
parasites.  They  inhabit  the  small  intestine,  often  in 
great  numbers,  and  commonly  produce  an  anemia  which 
is  often  severe  and  sometimes  fatal.  The  presence  of  a 
few,  however,  may  cause  slight,  if  any,  disturbance. 


Fig.  141. — Uncinaria  duodenalis:  a,  Male  (natural  size);  6,  female  (natural  size);  c,  male 
(enlarged);  d,  female  (enlarged);  e,  head;/,/,/,  eggs  (after  v.  Jaksch). 

Uncinaria  duodenalis  is  common  in  southern  Europe 
and  in  Egypt.  The  body  is  cylindric,  reddish  in  color, 
and  the  head  is  bent  sharply.  The  oral  cavity  has  six 
hook-like  teeth.  The  female  is  12  to  18  mm,  long  and 
the  tail  is  pointed.  The  male  is  8  to  10  mm.  long  and 
the  posterior  end  is  expanded  into  an  umbrella-Uke 
pouch,  the  caudal  bursa.  The  eggs  are  oval  and  have  a 
thin,  smooth,  transparent  shell.    As  they  appear  in  the 


4IO 


ANIMAL   PARASITES 


feces  the  protoplasm  is  divided  into  2,  4,  8,  or  more 
rounded  segments  (Fig.  141).  They  measure  32  to  38 
by  52  to  61  /I. 

Necator  americanus  is  very  common  in  subtropical 
America,  including  the  southern  part  of  the  United 


Fig.  142. — Ova  of  Necator  americanus  in  feces.    The  egg,  showing  three  cells,  is  a  lateral 
view  of  a  four-cell  stage  (X  23°)  (photographs  by  the  author). 

States  and  the  West  Indies.  In  Porto  Rico  90  per  cent, 
of  the  rural  population  is  infected.  Isolated  cases,  prob- 
ably imported,  have  been  seen  in  most  of  the  Northern 
States.  The  American  hook-worm  is  smaller  than  the 
Old  World  variety,  the  male  being  7  to  9  mm.  long,  the 


PHYLUM   NEMATHELMINTHES  411 

female  9  to  1 1  mm.  The  four  ventral  hook-like  teeth  are 
replaced  by  chitinous  plates.  There  are  also  differences 
in  the  caudal  bursa  of  the  male,  and  in  the  situation  of 
the  vulva  in  the  female.  The  ova  (Fig.  142)  resemble 
those  of  Uncinaria  duodenalis,  but  are  larger,  36  to  45 
by  64  to  75  ^. 

The  Hfe-history  of  the  two  worms  is  probably  the 
same.  The  ova  pass  out  with  the  feces,  and,  under  fa- 
vorable conditions  of  warmth  and  moisture,  devolop  an 
embryo  which  hatches  within  a  few  days.  The  resulting 
larvae  pass  through  a  stage  of  development  in  warm 
moist  earth,  growing  to  a  length  of  0.5  to  0.6  mm.,  and 
moulting  twice.  They  are  then  ready  to  infect  a  new 
host.  In  some  cases  they  probably  reach  the  host's 
intestine  by  way  of  the  mouth,  with  food  or  water;  but 
the  usual  route  is  probably  that  estabhshed  by  Loos. 
When  moist  earth  containing  the  larvae  comes  in  contact 
with  the  skin,  they  penetrate  into  the  subcutaneous 
tissues.  This  is  favored  by  retention  of  mud  between 
the  toes  of  those  who  go  barefooted.  When  the  larvae 
are  abundant  a  dermatitis  is  induced  ("ground  itch"). 
From  the  subcutaneous  tissue  they  pass  by  way  of 
lymph-  and  blood-streams  to  the  lungs.  Here  they  make 
their  way  into  the  smaller  bronchi,  are  carried  by  the 
bronchial  mucus  to  the  pharynx,  and  are  swallowed. 
They  thus  ultimately  reach  the  small  intestine,  where 
they  develop  into  mature  worms. 

The  diagnosis  of  hookworm  infection,  which  is  assum- 
ing increasing  importance  in  this  country,  must  rest  upon 
detection  of  ova  in  the  feces.  The  worms  themselves 
seldom  appear  except  after  thymol  and  a  cathartic.  A 
small  portion  of  the  feces,  diluted  with  water  if  nee- 


412 


ANIMAL   PARASITES 


essary,  is  placed  upon  a  slide 
and  the  larger  particles  re- 
moved. The  material  is  cov- 
ered and  searched  with  a  i6- 
mm.  objective.  A  higher 
power  may  rarely  be  neces- 
sary to  positively  identify  an 
egg,  but  should  not  be  used  as 
a  finder.  The  eggs  (see  Figs. 
135, 142)  are  nearly  always  typ- 
ical, showing  a  thin  but  very  dis- 
tinct shell,  a  clear  zone,  and  a 
finely  granular  segmented  pro- 
toplasm. A  light  spot,  repre- 
senting the  nucleus,  can  usually 
be  made  out  in  each  segment. 
After  having  once  been  seen 
the  eggs  are  not  easily  mis- 
taken. Larvae  are  not  found 
in  fresh  feces,  but  may  hatch 
within  twenty-four  to  forty- 
eight  hours,  and  are  then  dis- 
tinguished with  difficulty  from 
Strongyloides  intestinalis.  In 
severe  infections  eggs  may  be 
found  in  nearly  every  micro- 
scopic field ;  in  most  cases,  even 
though  comparatively  mild, 
they  can  be  found  on  the  first 


Fig.  143. — A,  Egg  of  Strongyloides  intestinalis  (parasitic  mother  worm)  found  in  stools 
of  case  of  chronic  diarrhea;  B,  Rhabditiform  larva  of  Strongyloides  intestinalis  from 
the  stools.    (William  Sydney  Thayer,  in  Journal  of  Experimental  Medicine.) 


PHYLUM   NEMATHELMINTHES  413 

slide  examined.  It  is  seldom  necessary  to  search  more 
than  half  a  dozen  slides.  From  the  estimate  of  Dock 
and  Bass  it  seems  probable  that  ova  will  average  at 
least  one  to  the  slide  if  ten  or  more  laying  females  are 
present  in  the  intestine.  Very  old  females  may  fail  to 
produce  eggs.  When  they  are  scarce,  some  method  of 
sedimenting  the  feces  may  be  tried  (see  p.  360). 

Pepper's  method  of  concentration  is  simple,  but  is  not 
applicable  to  other  ova  than  those  of  the  hookworm.  It 
is  best  first  to  sediment  the  feces.  A  layer  of  the  diluted 
feces  is  placed  on  a  slide  and  allowed  to  remain  for  some 
minutes.  The  sHde  is  then  gently  immersed  in  water. 
The  ova,  which  have  settled  to  the  bottom,  cling  to  the 
glass  and  are  not  washed  away  as  is  other  material. 
This  may  be  repeated  several  times  and  numerous  eggs 
collected. 

6.  Genus  Strongyloides.— (i)  Strongyloides  intes- 
tinalis. — Infection  with  this  worm  is  by  no  means  so  rare 
in  this  country  as  the  few  clinical  reports  would  indicate. 
It  is  apparently  widespread  in  the  Southern  States. 
It  is  very  common  in  subtropical  countries,  notably  in 
Italy  and  in  southern  China.  It  seems  probable  that 
the  parasite  is  the  cause  of  "Cochin  China  diarrhea," 
although  some  authorities  regard  it  as  harmless. 

The  adult  female,  which  reproduces  by  parthenogenesis 
and  is  about  2  mm.  long,  inhabits  the  upper  portion  of 
the  small  intestine,  but  neither  it  nor  the  ova  appear  in 
the  stool  unless  an  active  diarrhea  exists.  Ordinarily 
they  hatch  in  the  intestines,  and  when  infection  is 
severe  larvae  can  be  found  in  the  feces  in  large  num- 
bers. These  are  the  "rhabditiform  larvae,"  which  meas- 
ure 450  to  600  ;[^  by  16  to  20  /u  (Figs.  143,  144).     They 


414  ANIMAL   PARASITES 

are  actively  motile,  and  are  best  found  by  making  a 
small  depression  in  the  fecal  mass,  filling  it  with  water, 
and  keeping  in  a  warm  place  (preferably  an  incubator) 
for  twelve  to  twenty-four  hours.  The  larvae  will  col- 
lect in  the  water,  and  can  be  easily  found  by  trans- 
ferring a  drop  to  a  slide  and  examining  with  a  i6-nmi. 
objective.  The  inexperienced  worker  should  make  sure 
that  the  worms  move,  or  he  may  be  misled  by  the  veg- 
etable hairs  which  are  generally  present  in  the  feces. 


^ 


'^^:=^ 


Fig.  144. — Rhabditiform  larva  of  Strongyloides  intestinalis  in  feces  (X  150)  (photograph 
by  the  author). 

Certain  of  these  hairs  (notably  those  from  the  skin  of 
a  peach)  closely  resemble  small  worms  (see  page  356). 

Outside  the  body  the  rhabditiform  larvae  develop 
into  a  free-living,  sexually  differentiated  generation.  The 
young  of  this  generation  are  the  more  slender  "filariform 
larvae,"  which  constitute  the  infective  form.  Direct 
transformation  of  rhabditiform  into  filariform  larvae 
also  occurs.  Infection  takes  place  by  ingestion  or  by 
way  of  the  skin. 

7.  Genus    Trichinella.— (i)    Trichinella    spiralis.— 


PHYLUM   NEMATHELMINTHES 


415 


This  is  a  very  small  worm — adult  males,  1.5  to  1.6  by 
0.04  mm. ;  females,  3  to  4  by  0.06  mm.  Infection  in  man 
occurs  from  eating  of  insufficiently  cooked  pork,  which 
contains  encysted  larvae.  Ordinary  ''curing"  of  pork 
does  not  kill  them.  These  reach  maturity  in  the  small 
intestine.  Soon  after  copulation  the  males  die,  and  the 
females  penetrate  into  the  mucous  membrane.  They 
live  in  this  situation  about  six  weeks,  giving  birth  to 
great  numbers  of  youngs  averaging  as  high  as  15CX)  from 


Fig.  145. — ^Trichinella  spiralis  Oarva.,/  from  head  of  right  gastrocnemius  muscle;  seventh 
week  of  disease  (i6-mm.  objective;  eye-piece  4)  (Boston). 

a  single  female.  The  larvae  migrate  to  the  striated  mus- 
cles, chiefly  near  the  tendinous  insertions,  where  they 
grow  to  a  length  of  about  0.8  mm.,  and  finally  become 
encysted.  In  this  condition  they  may  remain  alive  and 
capable  of  developing  for  as  long  as  twenty-five  years. 

Trichiniasis  is  generally  accompanied  by  a  marked 
eosinophilia.  The  diagnosis  is  made  by  teasing  out  upon 
a  sHde  a  bit  of  muscle,  obtained  preferably  from  the  outer 
head  of  the  gastrocnemius,  the  insertion  of  the  deltoid, 
or  the  lower  portion  of  the  biceps.      The  coiled  larvae 


4l6  ANIMAL   PARASITES 

can  easily  be  seen  with  a  i6-mm.  objective  (Fig.  145). 
The  larvae  can  sometimes  be  found  in  the  blood  (see  p. 
305)  before  they  have  reached  their  final  resting-place 
in  the  muscles.  During  the  diarrheal  stage  adults  may 
be  present  in  the  feces,  and  are  found  by  diluting  with 
water  and  decanting  several  times  and  examining  the 
sediment  in  a  very  thin  layer  with  a  hand  lens. 

8.  Genus  Trichocephalus. — (i)  Trichocephalus  tri- 
chiurus. — This,  the  "whip-worm,"  is  3.5  to  5  cm.  long. 
Its  anterior  portion  is  slender  and  thread-like,  while 
the  posterior  portion  is  thicker  (Fig.  146).  It  is  widely 
distributed    geographically,    and    is    one    of    the   most 


Fig.  146. — ^Trichocephalus  trichiurus:  a,  Female;  b,  male  (natural  size)  (Heller). 

common  of  intestinal  parasites  in  this  country.  It  lives 
in  the  large  intestine,  especially  the  cecum,  with  its 
slender  extremity  embedded  in  the  mucous  membrane. 
Whip-worms  do  not,  as  a  rule,  produce  any  symptoms, 
although  gastro-intestinal  disturbances,  nervous  symp- 
toms, and  anemia  have  been  ascribed  to  them.  They, 
as  well  as  many  other  intestinal  parasites,  are  probably 
an  important  factor  in  the  etiology  of  appendicitis, 
typhoid  fever,  and  other  intestinal  infections.  The 
damage  which  they  do  to  the  mucous  membrane  favors 
bacterial  invasion. 

The  number  present  is  usually  small.     The  worms 
themselves  are  rarely  found  in  the  feces.    The  ova,  which 


PHYLUM   ARTHROPODA 


417 


are  not  often  abundant,  are  easily  recognized  with  the 
i6-mm.  objective.  Although  they  are  comparatively 
small,  their  appearance  is  striking.     They  are  brown, 


Fig.  147. — Ova  of  Trichocephalus  trichiurus  (Xsoo)  (photographs  by  the  author). 

ovoid  in  shape,  50  to  54  ^  long  by  about  23  (J.  wide,  and 
have  a  button-Hke  projection  at  each  end  (Fig.  147). 


PHYLUM  ARTHROPODA 

The  arthropoda  which  are  parasitic  to  man  belong  to 
the  classes  Arachnoidea  and  Insecta.  They  are  nearly 
all  external  parasites,  and  the  reader  is  referred  to  the 
standard  works  upon  diseases  of  the  skin  for  descriptions. 
The  several  species  of  the  louse  {Pediculus  capitis,  P. 
vestimenti,  P.  pubis) ,  the  itch  mite  {Sarcoptes  scahiei) ,  and 
the  small  organism  {Demodex  folliculorum)  which  lives 
in  the  sebaceous  glands,  especially  about  the  face,  are 
the  most  common  members  of  this  group. 

A  number  of  flies  may  deposit  their  ova  in  wounds  or  in 
such  of  the  body  cavities  as  they  can  reach,  and  the  re- 
sulting maggots  may  cause  intense  irritation.  Ova  may 
be  swallowed  with  the  food  and  the  maggots  appear  in  the 

27 


4i8 


ANIMAL   PARASITES 


feces.  Probably  most  important  is  the  "screw  worm," 
the  larva  of  Chrysomyia  macellaria,  infection  with  which 
is  not  rare  in  some  parts  of  the  United  States.  The  ova 
are  most  commonly  deposited  in  the  nasal  passages,  and 
the  larvae,  which  may  be  present  in  great  numbers, 
burrow  through  the  soft  parts, 
cartilage,  and  even  bone,  always 
with  serious  and  often  with  fatal 
results. 

A  few  cases  of  human  infec- 
tion with  Linguatula  serrata  have 
recently  been  reported  from  the 
Panama  Canal  Zone  and  from 
Brazil,  and  it  may  prove  to  be 
more  common  than  has  been  recog- 
nized. The  parasite  belongs  to 
the  class  Arachnoidea,  which  in- 
cludes spiders,  mites,  ticks,  etc.  It 
is  not  at  all  rare  in  Europe.  Re- 
lated species  are  common  in  cer- 
tain birds  in  North  America.  Man 
may  be  infected  with  either  adult 
or  larval  stages,  the  former  living 
in  the  nasal  and  accessory  passages, 
the  latter,  encysted,  in  the  inter- 
nal organs,  particularly  the  liver. 
The  larvae  may  be  found  in  the 
feces,  and,  because  of  their  serra- 
tions, may  be  mistaken  for  minute 
tapeworms  (Fig.  148).  They  are  white  in  color  and 
measure  about  4  to  6.5  mm.  long  and  0.9  to  1.5  mm. 
broad  at  the  widest  (anterior)  part. 


Fig.  148. — Larva  of  lingu- 
atula serrata  (de  Faria  and 
Travassos). 


CHAPTER  Vn 

MISCELLANEOUS  EXAMINATIONS 

PUS 

Pus  contains  much  granular  debris  and  numerous  more 
or  less  degenerated  cells,  the  great  majority  being  poly- 
morphonuclear leukocytes — so-called  "pus-corpuscles." 
Eosinophilic  leukocytes  are  common  in  gonorrheal  pus 
and  in  asthmatic  sputum.  Examination  of  pus  is  di- 
rected chiefly  to  detection  of  bacteria. 

When  very  few  bacteria  are  present,  culture  methods, 
which  are  outlined  in  Chapter  VIII,  must  be  resorted  to. 
When  considerable  mmibers  are  present,  they  can  be 
detected  and  often  identified  in  cover-glass  smears. 
Several  smears  should  be  made,  dried,  and  fixed,  as  de- 
scribed on  p.  466. 

One  of  these  should  be  stained  with  a  bacterial  stain. 
Lofiler's  methylene-blue  and  Pappenheim's  pyronin- 
methyl-green  are  especially  satisfactory  for  this  pur- 
pose. These  stains  are  apphed  for  one-half  minute 
to  two  minutes  or  longer,  without  heating;  the  prep- 
aration is  rinsed  in  water,  dried,  mounted,  and  examined 
with  an  oil-immersion  lens.  Another  smear  should  be 
stained  by  Gram's  method  (see  page  467).  These 
will  give  information  concerning  all  bacteria  which 
may  be  present,  and  frequently  no  other  procedure 
will  be  necessary  for  their  identification. 

419 


420 


MISCELLANEOUS   EXAMINATIONS 


The    most    common    pus-producing    organisms    are 
staphylococci  and  streptococci.    They  are  both  cocci,  or 


Fig.  149. — Staphylococcus  pyogenes  albus  from  an  abscess  of  the  parotid  gland  (Jakob). 

spheres,  their  average  diameter  being  about  i  (i.    Staphy- 
lococci are  commonly  grouped  in  clusters,  often  compared 


Fig.  1  so. — Streptococcus  pyogenes  from  a  case  of  empyema  Qakob). 

to  bunches  of  grapes   (Fig.   149).     There  are  several 
varieties  which  can  be  distinguished  only  in  cultures. 


PUS  421 

Streptococci  are  arranged  side  by  side,  forming  chains 
of  variable  length  (Fig.  150).  Sometimes  there  are  only 
three  or  four  individuals  in  a  chain;  sometimes  a  chain 
is  so  long  as  to  extend  across  several  microscopic  fields. 
Streptococci  are  more  virulent  than  staphylococci,  and 
are  less  commonly  met.  Both  are  Gram-positive. 
Their  cultural  characteristics  are  given  on  p.  475. 

Should  bacteria  resembHng  pneumococci  be  found, 
Buerger's  or  Smith's  method  for  capsules  (see  p.  72) 
should  be  tried.    When  these  are  not  available,  capsules 


Fig.  151. — Diplococcus  pneumoniae  from  ulcer  of  cornea  (obj.  one-twelfth  oil-immersion) 
(study  through  courtesy  of  Dr.  C.  A.  Oliver)  (Boston). 

can  usually  be  shown  by  the  method  of  Hiss.  The  dried 
and  fixed  smear  is  covered  with  a  stain  composed  of  5  c.c. 
saturated  alcoholic  solution  gentian-violet  and  95  c.c. 
distilled  water,  and  heated  until  steam  rises.  The  prep- 
aration is  then  washed  with  20  per  cent,  solution  of 
copper  sulphate,  dried,  and  mounted  in  Canada  balsam. 
Pneumococci  may  give  rise  to  inflammation  in  many 
locations  (see  p.  71).  When  they  form  short  chains, 
demonstration  of  the  capsule  is  necessary  to  distinguish 
them  from  streptococci. 


422  MISCELLANEOUS    EXAMINATIONS 

If  tuberculosis  be  suspected,  the  smears  should  be 
stained  by  one  of  the  methods  for  the  tubercle  bacillus 
(see  pp.  64  to  66),  or  guinea-pigs  may  be  inoculated. 
The  bacilli  are  generally  difficult  to  find  in  pus,  and 
bacteria-free  pus  would  suggest  tuberculosis. 

Gonococci,  when  t>T)ical,  can  usually  be  identified  with 
sufficient  certainty  for  clinical  purposes  in  the  smear 
stained  with  Loffler's  methylene-blue  or,  much  better, 
Pappenheim's  pyronin-methyl-green.     They  are  cofTee- 


Fig.  152. — Gonococci  in  urethral  pus  (McFarland). 

bean-shaped  cocci  which  lie  in  pairs  with  their  flat  sur- 
faces together  (Fig.  152).  They  lie  for  the  most  part 
within  pus-cells,  an  occasional  cell  being  filled  with  them, 
while  the  surrounding  cells  contain  few  or  none.  A  few 
are  found  outside  of  the  cells.  It  is  not  usual  to  find 
gonococci  when  many  other  bacteria  are  present,  even 
though  the  pus  is  primarily  of  gonorrheal  origin.  When- 
ever the  identity  of  the  organism  is  at  all  questionable, 
Gram's  method  should  be  tried.  In  rare  instances  it 
may  be  necessary  to  resort  to  cultures.    The  gonococcus 


PERITONEAL,   PLEURAL,   AND   PERICARDIAL   FLUIDS      423 

is  distinguished  by  its  failure  to  grow  upon  ordinary 
media  (see  p.  476) . 

Gonococci  are  generally  easily  found  in  pus  from  un- 
treated acute  and  subacute  gonorrheal  inflammations — 
conjunctivitis,  urethritis,  etc. — but  are  found  with  diffi- 
culty in  pus  from  chronic  inflammations  and  abscesses, 
and  in  urinary  sediments. 

PERITONEAL,  PLEURAL,  AND  PERICARDIAL  FLUIDS 

The  serous  cavities  contain  very  little  fluid  normally, 
but  considerable  quantities  are  frequently  present  as  a 
result  of  pathologic  conditions.  The  pathologic  fluids  are 
classed  as  transudates  and  exudates. 

Transudates  are  non-inflammatory  in  origin.  They 
contain  only  a  few  cells,  and  less  than  2.5  per  cent,  of 
albumin,  and  do  not  coagulate  spontaneously.  The 
specific  gravity  is  below  1.018.  Micro-organisms  are 
seldom  present. 

Exudates  are  of  inflammatory  origin.  They  are  richer 
in  cells  and  albumin,  and  tend  to  coagulate  upon  stand- 
ing. The  specific  gravity  is  above  i.oi 8.  The  amount  of 
albumin  is  estimated  by  Tsuchiya's  method,  after  di- 
luting the  fluid,  if  much  albumin  is  present.  A  mucin- 
like  substance,  called  serosomucin,  is  likewise  found  in 
exudates.  It  is  detected  by  acidifying  with  acetic  acid, 
when  a  white  cloudy  precipitate  results.  Some  transu- 
dates give  a  slight  turbidity  with  acetic  acid.  Bacteria 
are  generally  present  and  often  numerous.  When  none 
are  found  in  stained  smears  or  cultures,  tuberculosis  is 
to  be  suspected,  and  animal  inoculation  should  be  re- 
sorted to. 

Exudates  are  usually  classed  as  serous,  serofibrinous. 


424 


MISCELLANEOUS    EXAMINATIONS 


seropurulent,  purulent,  putrid,  and  hemorrhagic,  which 
terms  require  no  explanation.  In  addition,  chylous  and 
chyloid  exudates  are  occasionally  met,  particularly  in  the 
peritoneal  cavity.  In  the  chylous  form  the  milkiness  is 
due  mainly  to  the  presence  of  minute  fat-droplets,  and  is 
the  result  of  rupture  of  a  lymph-vessel,  usually  from 
obstruction  of  the  thoracic  duct.  Chyloid  exudates 
are  milky  chiefly  from  proteins  in  suspension,  or  fine 


% 

/^ 

r^Ph  «| 

'  m 

1 

1 

fl»       J 

Fig.  153. — Cytodiagnosis.  Polymorphonuclear  leukocytes  and  swollen  endothelial 
cells  from  acute  infectious  non-tuberculous  pleuritis  (Percy  Musgrave;  photo  by  L.  S. 
Brown). 


debris  from  broken-down  cells.  These  exudates  are  most 
frequently  seen  in  carcinoma  and  tuberculosis  of  the 
peritoneum. 

Cytodiagnosis. — This  is  diagnosis  from  a  differential 
count  of  the  cells  in  a  transudate  or  exudate,  particularly 
one  of  pleural  or  peritoneal  origin. 

The  fresh  fluid,  obtained  by  aspiration,  is  centrifugal- 
ized  for  at  least  five  minutes;  the  supernatant  liquid  is 
poured  off;  and  smears  are  made  from  the  sediment  and 


PERITONEAL,   PLEURAL,    AND   PERICARDIAL   FLUIDS      425 

dried  in  the  air.  The  smears  are  then  stained  with 
Wright's,  Harlow's  or  Jenner's  stain,  mounted,  and 
examined  with  an  oil-immersion  objective. 

Predominance  of  polymorphonicclear  leukocytes  (pus- 
corpuscles)  points  to  an  acute  infectious  process  (Fig. 
153).  These  cells  are  the  neutrophiles  of  the  blood. 
Eosinophiles  and  mast-cells  are  rare.  In  thin  smears 
they  are    easily  recognized,   the   cytoplasmic  granules 


Fig.  154. — Cytodiagnosis.  Lymphoid  cells  tmni  iK  ural  fluid;  case  of  tuberculous  pleuritis 
(Percy  Musgravc;  phuLo  by  L.  S.  Brown). 

often  staining  characteristically  with  polychrome-meth- 
ylene-blue-eosin  stains.  In  thick  smears,  upon  the  other 
hand,  they  are  often  small  and  shrunken,  and  may  be 
identified  with  difiiculty,  being  easily  mistaken  for  lym- 
phocytes. 

Predominance  of  lymphocytes  (Fig.  154)  generally  sig- 
nifies tuberculosis.  They  are  the  same  as  found  in  the 
blood.    The  cytoplasm  is  usually  scanty,  is  often  ragged. 


426  MISCELLANEOUS   EXAMINATIONS 

and  sometimes  is  apparently  absent  entirely.  Tuber- 
culous pleurisy  due  to  direct  extension  from  the  lung 
may  give  excess  of  polymorphonuclears  owing  to  mixed 
infection. 

Predominance  of  mesothelial  cells,  few  cells  of  any  kind 
being  present,  indicates  a  transudate  (Fig.  155).    These 


-Cytodiagnosis.     Mesothelial  cells  from  transudate  or  mechanical  effusion 
(Percy  Musgrave;  photo  by  L.  S.  Brown). 

cells  are  large,  with  relatively  abundant  cytoplasm,  and 
contain  one,  sometimes  two,  round  or  oval,  palely  stain- 
ing nuclei.  Mesothelial  cells  generally  predominate  in 
carcinoma,  but  are  accompanied  by  considerable  num- 
bers of  lymphocytes  and  red  blood-corpuscles.  Cancer 
cells  cannot  be  recognized  as  such,  although  the  presence 
of  mitotic  figures  would  suggest  malignant  disease. 

CEREBROSPINAL  FLUID 

Examination  of  the  fluid  obtained  by  lumbar  puncture 
has  of  recent  years  become  a  very  important  aid  in 
diagnosis. 


CEREBROSPINAL   FLUID  427 

1 .  Macroscopic  Examination. — The  amount  obtain- 
able varies  from  a  few  drops  to  100  c.c.  Normally, 
the  fluid  is  clear  and  limpid,  resembling  water.  The 
reaction  is  alkaline.  The  specific  gravity  is  1.003  to 
1.008.  Not  infrequently  it  is  tinged  with  fresh  blood 
from  a  punctured  vessel.  This  should  not  be  confused 
with  the  dull-red  or  brown  color  which  is  seen  in  hemor- 
rhagic conditions  Hke  intraventricular  and  subdural 
hemorrhage  and  hemorrhagic  meningitis.  When  the 
bleeding  is  extensive  and  recent  it  may  give  the  appear- 
ance of  practically  pure  blood. 

In  purulent  meningitis  the  fluid  may  exhibit  varying 
degrees  of  cloudiness,  from  slight  turbidity  to  almost 
pure  pus.  In  the  less  acute  stage  of  the  epidemic  form 
it  is  sometimes  quite  clear. 

After  standing  for  twelve  to  twenty-four  hours  the 
fluid  will  often  coagulate.  This  occurs  especially  in  the 
various  forms  of  meningitis,  rarely  in  non-inflammatory 
conditions.  In  tuberculosis  the  coagulum  is  usually  very 
delicate  and  cobweb-like  and  is  not  easily  seen. 

2.  Chemical  Examination. — Only  a  few  constituents 
are  of  clinical  importance. 

(i)  Globulin. — Traces  are  present  normally.  A 
notable  increase  occurs  in  acute  inflammations  and  in 
syphilis  and  parasyphilitic  affections.  The  two  tests 
for  globulin  which  follow  are  positive  in  93  to  95  per 
cent,  of  all  cases  of  paresis,  and  are,  therefore,  an  im- 
portant diagnostic  consideration.  When  acute  inflam- 
mation is  excluded,  they  run  practically  parallel  with 
-the  Wassermann  reaction  when  the  latter  is  applied  to 
the  spinal  fluid.  They  must  not  be  applied  to  fluid  con- 
taining blood,  owing  to  the  presence  of  serum-globulin. 


428  MISCELLANEOUS    EXAMINATIONS 

Noguchi's  Butyric  Acid  Test. — In  a  small  test-tube  take 
I  to  2  c.c.  of  the  fluid  and  5  c.c.  of  a  10  per  cent,  solution  of 
butyric  acid  in  normal  salt  solution.  The  original  test  calls 
for  one-tenth  these  quantities,  but  they  are  too  small  for 
convenient  manipulation.  Heat  to  boiling  and  immediately 
add  I  c.c.  of  normal  sodium  hydroxid  solution  and  boil  again 
for  a  few  seconds.  A  positive  reaction,  corresponding  to  a 
pathologic  amount  of  globulin,  varies  from  a  distinct  cloudi- 
ness to  a  heavy  flocculent  precipitate  which  generally  appears 
within  twenty  minutes,  but  may  be  delayed  for  two  hours.  A 
slight  opalescence  may  be  seen  in  normal  fluids. 

Ammonium  Sulphate  Test. — Globulin  is  precipitated  by 
strong  solutions  of  ammonium  sulphate.  Ross  and  Jones 
apply  the  test  after  the  manner  of  the  ring  tests  for  albumin 
in  the  urine.  In  a  test-tube  or  horismascope  take  a  few 
cubic  centimeters  of  a  saturated  solution  of  ammonium  sul- 
phate and  overlay  with  the  suspected  fluid.  In  the  presence 
of  an  excess  of  globulin,  a  clear-cut,  thin,  grayish-white  ring 
appears  at  the  zone  of  contact  of  the  two  fluids  within  five 
minutes  to  three  hours.  This  test  appears  to  be  only  very 
slightly  less  reliable  than  the  butyric  acid  test. 


(2)  Sugar. — The  normal  cerebrospinal  fluid  gives  a 
distinct  reaction  with  the  copper  tests  (see  pp.  138,  139), 
apparently  due  to  glucose.  A  number  of  writers  lay 
stress  upon  the  absence  of  this  reduction  in  meningitis. 
From  a  study  of  a  recent  series  of  cases,  Jacob  finds 
that:  (i)  No  reduction  of  copper  occurs  in  pyogenic 
meningitis  (pneumococcus,  streptococcus,  etc.)  or  in 
acute  meningococcic  meningitis;  (2)  reduction  occurs, 
but  may  be  diminished  in  tuberculosis  and  in  the  more 
chronic  cases  of  meningococcic  meningitis;  (3)  reduction 
is  normal  in  poliomyelitis. 


CEREBROSPINAL   FLUID  429 

(3)  Antimeningococcus-serum  Test. — Vincent  and 
other  French  investigators  have  developed  the  following 
test,  which  they  believe  to  be  specific  for  epidemic  cere- 
brospinal meningitis: 

To  a  few  cubic  centimeters  of  the  spinal  fluid,  which  has 
been  cleared  by  thorough  centrifugation,  are  added  a  few 
drops  of  antimeningoccocus  serum.  The  tube,  along  with 
a  control  tube  of  the  untreated  fluid,  is  then  placed  in  an 
incubator  at  52°  C.  for  a  few  hours.  A  positive  reaction  con- 
sists in  the  appearance  of  a  white  cloud.  The  test  is  said  to  be 
reliable  even  when  meningococci  cannot  be  found.  The 
serum  must  be  free  from  phenol  and  other  interfering  sub- 
stances. 

3.  Microscopic  Examination. — This  consists  in  a 
study  of  the  bacteria,  and  of  the  number  and  kind  of 
cells. 

(i)  Bacteria. — Tubercle  bacilli  can  be  found  in  the 
majority  of  cases  of  tuberculous  meningitis.  The  deli- 
cate coagulum  which  forms  when  the  fluid  is  allowed  to 
stand  in  a  cool  place  for  twelve  to  twenty-four  hours  will 
entagle  any  bacilli  which  may  be  present.  This  clot 
may  be  removed,  spread  upon  sUdes,  and  stained  by  one 
of  the  methods  already  given  (see  pp.  64  to  66).  If 
desired,  the  coagulum  may  be  treated  with  antiformin 
(see  p.  68) .  In  case  no  coagulum  forms,  the  fluid  should 
be  thoroughly  centrifugalized  and  the  sediment  stained, 
or,  if  much  protein  be  present,  it  may  be  coagulated  by 
heat,  precipitated  by  the  centrifuge,  and  treated  with 
.  antiformin.  It  may  be  necessary  to  examine  a  consider- 
able number  of  smears.  In  doubtful  cases  inoculation 
of  guinea-pigs  must  be  resorted  to. 


43©  MISCELLANEOUS   EXAMINATIONS 

The  Diplococcus  intracellularis  meningitidis  is  recog- 
nized as  the  cause  of  epidemic  cerebrospinal  fever,  and 
can  be  detected  in  the  cerebrospinal  fluid  of  most  cases, 
especially  those  which  run  ah  acute  course.  Cover-glass 
smears  from  the  sediment  should  be  stained  by  a  simple 


Fig.  156. — Diplococcus  intracellularis  meningitidis  in  leukocytes  (X2000)  fWrght  and 

Brown). 

bacterial  stain  and  by  Gram's  method.  The  meningo- 
coccus is  an  intracellular  diplococcus  which  often  cannot 
be  distinguished  from  the  gonococcus  in  stained  smears 
(Fig.  156).  It  also  decolorizes  by  Gram's  method.  The 
presence  of  such  a  diplococcus  in  meningeal  exudates 
is,  however,  sufficient  for  its  indentification  in  clinical 
work. 

Various  organisms  have  been  found  in  other  forms 
of  meningitis — the  pneumococcus  most  frequently,  the 


CEREBROSPINAL    FLUID 


431 


influenza  bacillus  (Fig.  157)  rarely.  In  some  cases 
no  micro-organisms  can  be  detected  even  by  culture 
methods. 

(2)  Cjrtology. — The  total  number  of  cells  is  counted 
with  the  hemacytometer.  Unna's  polychrome  methyl- 
ene-blue  or  a  solution  of  methyl-violet  or  other  nuclear 
dye  is  drawn  into  the  leukocyte  pipet  to  the  0.5  mark, 
and  the  fresh  spinal  fluid,  which  has  been  well  shaken,  is 


Fig.  157. — Influenza  bacilli  in  spinal  fluid.    Case  of  meningitis  (X  iooo>  (photou'raph  by 

the  author). 

drawn  up  to  the  mark  1 1 .  After  mixing,  a  drop  is  placed 
on  the  counting  slide  and  covered.  To  reduce  the  error 
arising  from  the  small  number  of  cells  present  it  is  neces- 
sary to  count  a  large  area  on  several  slides.  Normally, 
the  cells  rarely  exceed  5  or  7  per  c.mm.;  10  is  perhaps  the 
maximum.  The  differential  count  is  made  as  described 
on  page  277.  Ordinarily,  only  two  kinds  of  cells  are 
&een :  lymphocytes  and  polymorphonuclear  neutrophils. 
Lymphocytes  predominate  normally.    An  increase  in 


432  MISCELLANEOUS   EXAMINATIONS 

the  total  count,  together  with  predominance  of  lympho- 
cytes (over  70  per  cent.),  strongly  suggests  tuberculo- 
sis or  s>'philitic  disease  of  the  nervous  system,  such  as 
paresis.  It  has  been  observed  in  the  more  chronic  type 
of  epidemic  cerebrospinal  meningitis,  but  not  to  the 
same  extent. 

In  acute  meningitis  the  total  count  is  high  and  poly- 
morphonuclears prevail. 

ANIMAL  INOCULATION 

Inoculation  of  animals  is  one  of  the  most  reliable  means 
of  verifying  the  presence  of  certain  micro-organisms  in 
fluids  and  other  pathologic  material,  and  is  helpful  in 
determining  the  species  of  bacteria  which  have  been 
isolated  in  pure  culture. 

Clinically,  it  is  applied  most  frequently  to  demon- 
stration of  the  tubercle  bacillus  when  other  means  have 
failed  or  are  uncertain.  The  guinea-pig  is  the  most 
suitable  animal  for  this  purpose.  When  the  suspected 
material  is  fluid  and  contains  pus,  it  should  be  well 
centrifugalized,  and  i  or  2  c.c.  of  the  sediment  injected 
by  means  of  a  large  h^'podermic  needle  into  the  peri- 
toneal ca\-ity  or  underneath  the  loose  skin  of  the  groin. 
Fluids  from  which  no  sediment  can  be  obtained  must 
be  injected  directly  into  the  peritoneal  cavity,  since 
at  least  10  c.c.  are  required,  which  is  too  great  an 
amount  to  inject  hvpodermically.  Solid  material  should 
be  placed  in  a  pocket  made  by  snipping  the  skin  of 
the  groin  with  scissors,  and  freeing  it  from  the  under- 
lying tissues  for  a  short  distance  around  the  opening. 
When  the  intraperitoneal  method  is  selected,  several 
animals  must  be  inoculated,  since  some  are  likely  to  die 


THE   MOUTH  433 

from  peritonitis  caused  by  other  organisms  before  the 
tubercle  bacillus  has  had  time  to  produce  its  character- 
istic lesions. 

The  animals  should  be  killed  at  the  end  of  six  or  eight 
weeks,  if  they  do  not  die  before  that  time,  and  a  careful 
search  should  be  made  for  the  characteristic  pearl-gray 
or  yellow  tubercles  scattered  over  the  peritoneum  and 
through  the  abdominal  organs,  particularly  the  spleen 
and  Uver,  and  for  caseous  inguinal  and  retroperitoneal 
lymph-glands.  The  tubercles  and  portions  of  the  caseous 
glands  should  be  crushed  between  two  slides,  dried,  and 
stained  for  tubercle  bacilli.  The  bacilli  are  difficult  to 
find  in  the  caseous  material. 

THE  MOUTH 
Micro-organisms  are  always  present  in  large  numbers. 
Among  these  is  Leptothrix  buccalis  (Fig.  158),  which  is 


Fig.  158. — Gingival  deposit  (unstained):  a.  Squamous  epithelial  cells;  b,  leukocytes;  c, 
bacteria;  d,  Leptothrix  buccalis  (Jakob). 

especially  abundant  in  the  crypts  of  the  tonsils  and  the 
tartar  of  the  teeth.     The  whitish  patches  of  Pharyn- 

28 


434 


MISCELLANEOUS    EXAMINATIONS 


gomycosis  leplothrica  are  largely  composed  of  these  fungi. 
They  are  slender,  segmented  threads,  which  generally, 
but  not  always,  stain  violet  with  Lugol's  solution,  and 
are  readily  seen  with  a  4-mm.  objective.  At  times  they 
are  observed  in  the  sputum  and  stomach  fluid.  In  the 
former  they  might  be  mistaken  for  elastic  fibers;  in  the 
latter,  for  Boas-Oppler  bacilli.  In  either  case,  the  re- 
action with  iodin  will  distinguish  them. 


Fig.  159. — Thrush  fungus  (Endomyces  albicans)  (KoUe  and  Wassermann). 


Thrush  is  a  disease  of  the  mouth  seen  most  often  in 
children,  and  characterized  by  the  presence  of  white 
patches  upon  the  mucous  membrane.  It  is  caused  by  the 
thrush  fungus,  Endomyces  albicans.  When  a  bit  from  one 
of  the  patches  is  pressed  out  between  a  slide  and  cover 
and  examined  with  a  4-mm.  objective,  the  fungus  is  seen 
to  consist  of  a  network  of  branching  segmented  hyphae 
with  numerous  spores,  both  within  the  hyphae  and  in  the 
meshes  between  them  (Fig.  159).  The  meshes  also  con- 
tain leukocytes,  epithelial  cells,  and  granular  debris. 


THE  MOUTH  435 

Acute  pseudomembranous  inflammations,  which  occur 
chiefly  upon  the  tonsils  and  nasopharynx,  are  generally 
caused  by  the  diphtheria  bacillus,  but  may  result  from 
streptococcic  infection.  In  many  cases  diphtheria 
bacilli  can  be  demonstrated  in  smears  made  from  the 
membrane  and  stained  with  Loffler's  methylene-blue  or 
2  per  cent,  aqueous  solution  of  methyl-green.    They  are 


m 

M 

m 

fc^£^sfi3 

pppwp 

>  . 

9 

' . 

/ 

^^-^' 

->:^. 

^  -'i-^^J', 

\ 

<Xs 

' 

> 

c 

V. 

•'  \ 

\ 

^  ■ 

t 

.-.' 

^J      ^        V,    JK    V               , 

■  *    .--  ■ 

_i  ;c: 

1 

. 

*  ^'. 

.   ** 

-  > 

• 

^-^        >•:-     : 

■  -•  / . 

v>« 

'^  V' .' . 

■' 

■'     ■'  ■:■:   \'^ 

^. 

■*  ■■ 

'              '          \ 

Fig.  i6o. — Bacillus  diphtheriae  stained  with  methyl-green;  culture  from  throat  (Xiooo) 
(photograph  by  the  author). 


straight  or  curved  rods,  which  vary  markedly  in  size 
and  outhne,  and  stain  very  irregularly.  A  characteristic 
form  is  a  palely  tinted  rod  with  several  deeply  stained 
granules  (metachromatic  bodies),  or  with  one  such 
granule  at  each  end  (Fig.  i6o).  They  stain  by  Gram's 
.method.  It  is  generally  necessary,  and  always  safer,  to 
make  a  culture  upon  blood-serum,  incubate  for  twelve 
hours,  and  examine  smears  from  the  growth. 


436  MISCELLANEOUS   EXAMINATIONS 

Neisser's  stain  has  long  been  the  standard  diflferential 
stain  for  the  diphtheria  bacillus.  It  colors  the  bodies  of  the 
bacilli  brown  and  the  metachromatic  bodies  blue. 

1.  Make  films  and  fix  as  usual. 

2.  Apply  the  following  solution,  freshly  filtered,  for  about 
one-half  minute: 

Methylene-blue o.i  gm.; 

Alcohol  (96  per  cent.) 2.0  c.c; 

Glacial  acetic  acid 5.0    " 

Distilled  water 95.0    " 

3.  Rinse  in  water. 

4.  Apply  a  saturated  aqueous  solution  of  Bismarck  brown 
one-half  minute. 

5.  Rinse,  dry,  and  mount. 

Pender's  Stain. — This  new  stain  is  preferred  by  many  to 
Neisser's: 

Toluidin  blue  (Gruebler) 0.02  gm.; 

Glacial  acetic  acid i.oo  c.c; 

Absolute  alcohol 2.00  " 

Distilled  water  to 100.00  " 

Cover  the  fixed  fihn  with  the  stain;  turn  the  cover-glass 
over  and  examine  as  a  hanging-drop  preparation.  Diph- 
theria bacilli  are  blue,  with  red  granules. 

Vincent's  angina  is  a  pseudomembranous  and  ulcer- 
ative inflammation  of  mouth  and  pharynx,  which  when 
acute  may  be  mistaken  for  diphtheria,  and  when  chronic 
is  very  apt  to  be  mistaken  for  syphilis.  Stained  smears 
from  the  ulcers  or  membrane  show  large  numbers  of 
spirochetes  and  "fusiform  bacilli,"  giving  a  striking  and 
characteristic  picture  (Fig.  161).  The  "bacillus"  is 
spindle  shaped,  more  or  less  pointed  at  the  ends,  and 


THE  MOUTH  437 

about  6  to  12  (I  long.  The  spirillum  is  a  very  slender, 
wavy  thread,  about  30  to  40  //  long,  and  stains  feebly. 
Diluted  analin-gentian- violet  makes  a  satisfactory  stain. 
With  methylene-blue  the  palely  staining  spirillum  may 
easily  be  overlooked.     Further  description  is  given  on 

P-  374. 

Tuberculous  ulcerations  of  mouth  and  pharynx  can 
generally  be  diagnosed  from  curetings  made  after  careful 


■ 

^-^  ^ 

-            -:        -K-  « 

/ 

Fig.  161. — SpirochaJta  \incenti  from  case  of  ulcerative  stomatitis  stained  with  gentian- 
violet  (X1200). 

cleansing  of  the  surface.  The  curetings  are  well  rubbed 
between  slide  and  cover,  and  the  smears  thus  made  are 
dried,  fixed,  and  stained  for  tubercle  bacilli.  Since  there 
is  much  danger  of  contamination  from  tuberculous  spu- 
tum, the  presence  of  tubercle  bacilli  is  significant  only  in 
proportion  to  the  thoroughness  with  which  the  ulcer  was 
cleansed.    The  diagnosis  is  certain  when  the  bacilli  are 


438  MISCELLANEOUS    EXAMINATIONS 

found  within  groups  of  cells  which  have  not  been  dis- 
sociated in  making  the  smears. 

THE  EYE 

Staphylococci,  pneumococci,  and  streptococci  are  prob- 
ably the  most  common  of  the  bacteria  to  be  found  in  non- 
specific conjunctivitis  and  keratitis.  Serpiginous  ulcer 
of  the  cornea  is  generally  associated  with  the  pneumococ- 
cus  (see  Fig.  151). 

The  usual  cause  of  acute  infectious  conjunctivitis 
("pink-eye"),  especially  in  cities,  seems  to  be  the  Koch- 
Weeks  bacillus.     This  is  a  minute,  slender  rod,  which 


•'"% 


w 


«> 


\'/ 


/ 


Fig.  162. — Conjunctival  secretion  from  acute  contagious  conjunctivitis;  polynuclear 
leukocytes  with  the  bacillus  of  Weeks;  P,  phagocyte  containing  bacillus  of  Weeks  (one- 
twelfth  oil-immersion,  ocular  iii)  (Morax). 

lies  within  and  between  the  pus-corpuscles  (Fig.  162), 
and  is  negative  to  Gram's  stain.  In  smears  it  cannot  be 
distinguished  from  the  influenza  bacillus,  although  its 
length  is  somewhat  greater. 

The  diplobacillus  of  Morax  and  Axenfeld  gives  rise 
to  an  acute  or  chronic  blepharoconjunctivitis  without 
follicles  or  membrane,  for  which  zinc  sulphate  seems  to  be 
a  specific.     It  is  widely  distributed  geographically  and 


THE   EYE  439 

is  common  in  many  regions.  The  organism  is  a  short, 
thick  diplobacillus,  is  frequently  intracellular,  and  is 
Gram-negative  (Fig.  163).  A  delicate  capsule  can  some- 
times be  made  out. 

Early  diagnosis  of  gonorrheal  ophthalmia  is  extremely 
important,  and  can  be  made  with  certainty  only  by  detec- 
tion of  gonococci  in  the  discharge.  They  are  easily  found 
in  smears  from  untreated  cases.     After  treatment  is 


Fig.  163. — The  diplobacillus  of  Moraz  and  Axenfeld  (from  a  preparation  by  Dr.  Harold 

GifFord). 

begun  they  soon  disappear,  even  though  the  discharge 
continues. 

Pseudomembranous  conjunctivitis  generally  shows 
either  streptococci  or  diphtheria  bacilli.  In  diagnosing 
diphtheric  conjunctivitis,  one  must  be  on  his  guard 
against  the  Bacillus  xerosis,  which  is  a  frequent  inhabit- 
ant of  the  conjunctival  sac  in  healthy  persons,  and  which 
is  identical  morphologically  with  the  diphtheria  bacillus. 


440  MISCELLANEOUS   EXAMINATIONS 

Hence  the  clinical  picture  is  more  significant  than  the 
microscopic  findings. 

Various  micro-organisms — bacteria,  molds,  protozoa — 
have  been  described  in  connection  with  trachoma,  but  the 
specific  organism  of  the  disease  is  not  definitely  known. 

Herbert  has  called  attention  to  the  abundance  of 
eosinophihc  leukocytes  in  the  discharge  of  vernal  catarrh. 
He  regards  their  presence  in  considerable  numbers  as 
very  helpful  in  the  diagnosis  of  this  disease. 

THE  EAR 

By  far  the  most  frequent  exciting  causes  of  acute  otitis 
media  are  the  pneumococcus  and  the  streptococcus.  The 
finding  of  other  bacteria  in  the  discharge  generally  indi- 
cates a  secondary  infection,  except  in  cases  complicating 
infectious  diseases,  such  as  typhoid  fever,  diphtheria,  and 
influenza.  Discharges  which  have  continued  for  some 
time  are  practically  always  contaminated  with  the 
staphylococcus.  The  presence  of  the  streptococcus 
should  be  a  cause  of  uneasiness,  since  it  much  more 
frequently  leads  to  mastoid  disease  and  meningitis  than 
does  the  pneumococcus.  The  staphylococcus,  bacillus 
of  Friedlander,  colon  bacillus,  and  Bacillus  pyocyaneus 
may  be  met  in  chronic  middle-ear  disease. 

In  tuberculous  disease  the  tubercle  bacillus  is  present  in 
the  discharge,  but  its  detection  offers  some  difficulties.  It 
is  rarely  easy  to  find,  and  precautions  must  always  be 
taken  to  exclude  the  smegma  and  other  acid-fast  bacilli 
(see  p.  69),  which  are  especially  liable  to  be  present  in 
the  ear.  Rather  striking  is  the  tendency  of  old  squamous 
cells  to  retain  the  red  stain,  and  fragments  of  such  cells 
may  mislead  the  unwary. 


MILK  441 

PARASITIC  DISEASES  OF  THE  SKIN 

Favus,  tinea  versicolor,  and  the  various  forms  of  ring- 
worm are  caused  by  members  of  the  fungus  group.  To 
demonstrate  them,  a  crust  or  a  hair  from  the  affected 
area  is  softened  with  a  few  drops  of  20  per  cent,  caustic 
soda  solution,  pressed  out  between  a  slide  and  cover,  and 
examined  with  a  4-mm.  objective.  They  consist  of  a 
more  or  less  dense  network  of  hyphae  and  numerous 
round  or  oval  refractive  spores.  The  cuts  in  standard 
works  upon  diseases  of  the  skin  will  aid  in  dififerentiating 
the  members  of  the  group. 

MILK 

A  large  number  of  analyses  of  human  and  cows'  milk 
are  averaged  by  Holt  as  follows,  Jersey  milk  being  ex- 
cluded because  of  its  excessive  fat: 

Human  Milk.  Cows'  Milk. 

Normal  variations,  Average,  Average, 

per  cent.  per  cent.  per  cent. 

Fat 3.00    to     5.00  4.00  3.50 

Sugar 6.00    to     7.00  7.00  4.30 

Proteins i.oo    to     2.25  1.50  4.00 

Salts 0.18    to     0.25  0.20  0.70 

Water 89.82    to  85.50  87.30  87.50 

100.00       100.00  100.00  100.00 

The  reaction  of  human  milk  is  slightly  alkaline;  of 
cows',  neutral  or  shghtly  acid.  The  specific  gravity  of 
each  is  bout  1.028  to  1.032.  Human  milk  is  sterile  when 
secreted,  but  derives  a  few  bacteria  from  the  lacteal 
ducts.  Cows'  milk,  as  usually  sold,  contains  large  num- 
bers of  bacteria,  the  best  milk  rarely  containing  fewer 
than  10,000  per  cubic  centimeter.  Microscopically, 
human  milk  is  a  fairly  homogeneous  emulsion  of  fat, 
and  is  practically  destitute  of  cellular  elements.     Any 


442 


MISCELLANEOUS   EXAMINATIONS 


notable  number  of  leukocytes  indicates  infection  of  the 
mammary  gland. 

Chemical  examination  of  milk  is  of  great  value  in  solv- 
ing the  problems  of  infant  feeding.   The  sample  examined 


ex. 

0___IO| 

'-X9 

2^-8 

3_L  r 

4_|_« 
5  Xs 

6_i-4 
7_|_3 
8-1-2 
9-J_« 
10_L-0 


3 


Fig.  164. — Holt's  milk-testing  apparatus. 

should  be  the  middle  milk,  or  the  entire  quantity  from 
one  breast.  The  fat  and  protein  can  be  estimated 
roughly,  but  accurately  enough  for  many  clinical  pur- 
poses by  means  of  Holt's  apparatus,  which  consists  of 
a  lo-c.c.  cream  gage  and  a  small  hydrometer  (Fig.  164). 


MILK 


443 


The  cream  gage  is  filled  to  the  o  mark  with  milk,  allowed 
to  stand  for  twenty-four  hours  at  room  temperature, 
and  the  percentage  of  cream  then  read  off.  The  per- 
centage of  fat  is  three-fifths  that  of  the  cream.  The 
protein  is  then  approximated  from  a  consideration  of 
the  specific  gravity  and  the  percentage  of  fat.  The  salts 
and  sugar  very  seldom  vary  sufficiently  to  affect  the 
specific  gravity,  hence  a  high  specific  gravity  must  be 
due  to  either  an  increase  of  protein  or  decrease  of  fat,  or 
both,  and  vice  versa.  With  normal  specific  gravity  the 
protein  is  high  when  the  fat  is  high,  and  vice  versa. 
The  method  is  not  accurate  with  cows' 
milk. 

For  more  accurate  work  the  following 
methods,  appUcable  to  either  human  or 
cows'  milk,  are  simple  and  satisfactory: 

Fat. — Lefmann-Beam  Method. — This 
is  essentially  the  widely  used  Babcock 
method,  modified  for  the  small  quanti- 
ties of  milk  obtainable  from  the  human 
mammary  gland.  The  apparatus  con- 
sists of  a  special  tube  which  fits  the 
aluminum  shield  of  the  medical  centri- 
fuge (Fig.  165)  and  a  5-c.c.  pipet.  Owing 
to  its  narrow  stem,  the  tube  is  difficult  to 
fill  and  to  clean.  Exactly  5  c.c.  of  the 
milk  are  introduced  into  the  tube  by  means  of  the  pipet, 
and  I  c.c.  of  a  mixture  of  equal  parts  of  concentrated 
hydrochloric  acid  and  amyl-alcohol  is  added  and  well 
mixed.  The  tube  is  filled  to  the  o  mark  with  concentrated 
sulphuric  acid,  adding  a  few  drops  at  a  time  and  agitating 
constantly.     This  is  revolved  in  the  centrifuge  at  1000 


Fig.  165. — Centri- 
fuge tube  for  milk 
analysis. 


444  MISCELLANEOUS   EXAMINATIONS 

revolutions  a  minute  for  three  minutes,  or  until  the  fat 
has  separated.  The  percentage  is  then  read  off  upon  the 
stem,  each  small  division  representing  0.2  per  cent,  of  fat. 
Proteins. — T.  R.  Boggs^  Modification  of  the  Esbach 
Method. — This  is  applied  as  for  urinary  albumin  (see 
p.  134),  substituting  Boggs'  reagent  for  Esbach's.  The 
reagent  is  prepared  as  follows: 

(i)  Phosphotungstic  acid 25  gm.; 

Distilled  water 125  c.c; 

(2)  Concentrated  hydrochloric  acid 25   " 

Distilled  water 100   " 

When  the  phosphotungstic  acid  is  completely  dissolved, 
mix  the  two  solutions.  This  reagent  is  quite  stable  if 
kept  in  a  dark  glass  bottle. 

Before  examination,  the  milk  should  be  diluted  accord- 
ing to  the  probable  amount  of  protein,  and  allowance 
made  in  the  subsequent  reading.  For  human  milk  the 
optimum  dilution  is  i:  10;  for  cows'  milk,  i:  20.  Dilu- 
tion must  be  accurate. 

Lactose. — The  protein  should  first  be  removed  by 
acidifying  with  acetic  acid,  boiling,  and  filtering.  The 
copper  methods  may  then  be  used  as  for  glucose  in  the 
urine  (see  p.  142);  but  it  must  be  borne  in  mind  that 
lactose  reduces  copper  more  slowly  than  glucose,  and 
longer  heating  is,  therefore,  required;  and  that  10  c.c.  of 
Fehling's  solution  (or  25  c.c.  of  Benedict's)  are  equiva- 
lent to  0.0676  gm,  lactose  (as  compared  with  0.05  gm. 
glucose) . 

Detection  of  Preservatives. — Formalin  is  the  most 
common  preservative  added  to  cows'  milk,  but  boric 
acid  is  also  used. 


SYPHILITIC   MATERIAL  445 

To  detect  formalin,  add  a  few  drops  of  dilute  ferric 
chlorid  solution  to  a  few  cubic  centimeters  of  the  milk, 
and  run  the  mixture  gently  upon  the  surface  of  some 
strong  sulphuric  acid  in  a  test-tube.  If  formaldehyd  be 
present,  a  bright  red  ring  will  appear  at  the  line  of  con- 
tact of  the  fluids.  This  is  not  a  specific  test  for  for- 
maldehyd, but  nothing  else  likely  to  be  added  to  the 
milk  will  give  it. 

To  detect  boric  acid,  Goske's  method,  as  used  by  the 
Chicago  Department  of  Health,  is  simple  and  satis- 
factory: Mix  2  c.c.  of  concentrated  hydrochloric  acid 
with  20  c.c.  of  the  milk  and  place  in  a  50-c.c.  beaker. 
In  this  suspend  a  long  strip  of  turmeric  paper  (2  cm. 
wide),  so  that  its  end  reaches  to  the  bottom  of  the 
beaker.  Allow  to  remain  about  half  an  hour.  The 
liquid  will  rise  by  capillarity,  and  if  boric  acid  be  present 
a  red-brown  color  will  appear  at  the  junction  of  the 
moist  and  dry  portions  of  the  paper.  If  this  is  touched 
with  ammonia,  a  bluish-green  slate  color  develops.  A 
rough  idea  of  the  amount  of  boric  acid  may  be  had  by 
comparing  the  depth  of  color  with  that  produced  by 
boric  acid  solutions  of  known  strength. 

SYPHILITIC   MATERIAL 

In  1905  Schaudinn  and  Hoffmann  described  the  occur- 
rence of  a  very  slender,  spiral  micro-organism  in  the 
lesions  of  syphilis.  This  they  named  Spirochceta  pallida, 
because  of  its  low  refractive  power  and  the  difficulty 
with  which  it  takes  up  staining  reagents.  The  name 
was  later  changed  to  Treponema  pallidum.  Its  etiologic 
relation  to  syphilis  is  now  universally  admitted.  It  is 
found  in  primary,  secondary,  and  tertiary  lesions,  but 


446  MISCELLANEOUS   EXAMINATIONS 

is  not  present  in  the  latter  in  sufficient  numbers  to  be 
of  value  in  diagnosis. 

Treponema  pallidum  is  an  extremely  slender,  spiral, 
motile  thread,  with  pointed  ends.  There  is  a  flagellum 
at  each  end,  but  it  is  not  seen  in  ordinary  preparations. 
The  organism  varies  considerably  in  length,  the  average 
being  about  7  /M,  or  the  diameter  of  a  red  blood-corpuscle; 
and  it  exhibits  three  to  twelve,  sometimes  more,  spiral 
curves,  which  are  sharp  and  regular  and  resemble  the 


Fig.  166. — Treponema  pallidum  (Xiooo)  (Leitz  ^  oil-immersion  objective  and  Leitz 
dark -ground  condenser). 

curves  of  a  corkscrew  (Figs.  113,  166,  167).  It  is  so 
delicate  that  it  is  difficult  to  see  even  in  well-stained 
preparations;  a  high  magnification  and  careful  focusing 
are,  therefore,  required.  Upon  ulcerated  surfaces  it  is 
often  mingled  with  other  spiral  micro-organisms,  which 
adds  to  the  difficulty  of  its  detection.  The  most  notable 
of  these  is  Spirochceta  refringens,  described  on  p.  375. 

Treponema  pallidum  is  most  easily  demonstrated  in 
chancres  and  mucous  patches,  although  the  skin  lesions 


SYPHILITIC   MATERIAL  447 

— papules,  pustules,  roseolous  areas — often  contain  large 
numbers.  Tissue-juice  from  the  deeper  portions  of  the 
lesions  is  the  most  favorable  material  for  examination, 
because  the  organisms  are  commonly  more  abundant 
than  upon  ulcerated  surfaces  and  are  rarely  accompanied 
by  other  micro-organisms.  After  cleansing,  the  surface 
is  gently  scraped  with  a  curet  or  rubbed  briskly  with  a 
swab  of  cotton  or  gauze.  In  a  few  moments  serum  will 
exude.     The  rubbing  should  not  be  so  vigorous  as  to 


Fig.  167. — Treponema  pallidum  and  Spirochasta  refringens  (X1200)  (Leitz  oil-immersion 

objective). 

bring  the  blood,  because  the  corpuscles  may  hide  the 
treponema.  Very  thin  cover-glass  smears  are  then  made 
from  the  serum. 

Staining  Methods. — Giemsa's  stain  is  probably  the  most 
widely  used.  It  is  best  purchased  ready  prepared.  Smears 
are  fixed  in  absolute  alcohol  for  fifteen  minutes.  Ten  drops 
of  the  stain  are  added  to  10  c.c.  of  faintly  alkaline  distilled 
water  (i  drop  of  a  i  per  cent,  solution  of  potassium  carbonate 
to  10  c.c.  of  the  water),  and  the  fixed  smear  is  immersed  in 
this  diluted  stain  for  one  to  three  hours  or  longer.    It  is  then 


448  MISCELLANEOUS   EXAMINATIONS 

rinsed  in  distilled  water,  dried,  and  mounted.  In  well-stained 
specimens  Treponema  pallidum  is  reddish;  most  other  micro- 
organisms, bluish.  More  intense  staining  may  be  obtained 
by  gently  warming  the  stain. 

Wright's  blood-stain,  used  in  the  manner  already  described 
(see  p.  268)  except  that  the  diluted  stain  is  allowed  to  act 
upon  the  film  for  fifteen  minutes,  gives  good  results. 

Silver  Method. — The  silver  impregnation  method  has  long 
been  used  for  tissues.    Stein  has  applied  it  to  smears  as  follows : 

1.  Dry  the  films  in  the  incubator  at  37°  C.  for  three  or 
four  hours. 

2.  Immerse  in  10  per  cent,  silver  nitrate  solution,  in  diffuse 
daylight,  for  some  hours,  until  the  preparation  takes  on  a 
metallic  luster. 

3.  Wash  in  water,  dry,  and  mount. 

The  organisms  are  black  against  a  brownish  backgrovmd. 

India-ink  Method. — A  small  drop  of  India-ink  of  good 
grade  (Glinther  and  Wagner's  "Chin-Chin  liquid  pearl" 
or  Griibler's  "nach  Burri"  recommended)  is  mixed  on  a 
slide  with  i  or  2  small  drops  of  serum  from  the  suspected 
lesion.  The  mixture  is  then  spread  over  the  slide  and 
allowed  to  dry.  After  drying,,  it  is  examined  with  an  oil- 
immersion  lens.  Micro-organisms,  including  Treponema  pal- 
lidum, appear  clear  white  on  a  brown  or  black  background, 
much  as  they  do  with  the  dark  ground  condenser  (see  Fig. 
166).  If  desired,  the  mixture  of  ink  and  serum  may  be 
covered  with  a  cover-glass  and  examined  in  the  fresh  state, 
the  living  organisms  being  thus  demonstrated.  Because  of 
its  extreme  simplicity  this  method  has  been  favorably  re- 
ceived. It  cannot,  however,  be  absolutely  relied  upon, 
since,  as  has  been  pointed  out,  many  India-inks  contain 
wavy  vegetable  fibrils  which  might  easily  mislead  a  begin- 
ner, and  sometimes,  indeed,  even  an  experienced  worker. 
Instead  of  India-ink,  collargol,  diluted  i  :  20  w'th  water 
and  thoroughly  shaken,  has  been  recommended. 


SEMEN  449 

Dark  ground  illumination  (see  p.  22)  may  be  used  to 
study  the  living  organisms  in  fresh  tissue  juices.  This  ofifers 
a  satisfactory  m-sans  of  diagnosis,  but  since  the  instrument  is 
expensive  the  practitioner  will  rely  upon  one  or  more  of  the 
staining  methods  just  eniunerated. 

Method  of  Oppenheim  and  Sachs. — ^Very  thin  air-dried 
films  are  stained  for  from  thirty  seconds  to  three  minutes 
with  phenol-gentian-violet  (saturated  alcoholic  solution  of 
gentian-violet,  10  c.c;  5  per  cent,  phenol,  90  c.c).  Previous 
fixation  is  not  necessary. 

SEMEN 

Absence  of  spermatozoa  is  a  more  common  cause  of 
sterility  than  is  generally  recognized.  In  some  cases 
they  are  present,  but  lose  their  motility  immediately 
after  ejaculation. 

Semen  should  be  kept  warm  until  examined.  When  it 
must  be  transported  any  considerable  distance,  the 
method  suggested  by  Boston  is  convenient:  The  fresh 
semen  is  placed  in  a  small  bottle,  to  the  neck  of  which  a 
string  is  attached.  This  is  then  suspended  from  a  button 
on  the  trousers,  so  that  the  bottle  rests  against  the  skin  of 
the  inguinal  region.  It  may  be  carried  in  this  way  for 
hours.  When  ready  to  examine,  place  a  small  quantity 
upon  a  warmed  slide  and  apply  a  cover.  The  spermat- 
ozoa are  readily  seen  with  a  4-mm.  objective  (see  Fig. 
58).   Normally,  they  are  abundant  and  in  active  motion. 

Detection  of  semen  in  stains  upon  clothing,  etc.,  is 
often  important.  The  finding  of  spermatozoa,  after 
soaking  the  stain  for  an  hour  in  normal  salt  solution  or 
dilute  alcohol  and  teasing  in  the  same  fluid,  is  absolute 
proof  that  the  stain  in  question  is  semen,  although  it  is 
not  possible  to  distinguish  human  semen  from  that  of  the 

29 


45° 


MISCELLANEOUS    EXAMINATIONS 


lower  animals  in  this  way.    A  little  eosin  added  to  the 
fluid  will  bring  the  spermatozoa  out  more  clearly. 

Florence's  Reaction. — The  suspected  material  is  soft- 
ened with  water,  placed  upon  a  sUde  with  a  few  drops 
of  the  reagent,  and  examined  at  once  with  a  medium 
power  of  the  microscope.  If  the  material  be  semen, 
there  will  be  found  dark-brown  crystals  (Fig.  i68)  in  the 
form  of  rhombic  platelets  resembUng  hemin  crystals,  or 


Fig.  i68  — Seminal  crystals  (medium  size)  (Xyso)  from  a  stain  on  clothing.  A  single 
thread  J  inch  long  was  used  in  the  test,  the  stain  being  three  years  and  four  months  old 
(Peterson  and  Haines). 

of  needles,  often  grouped  in  clusters.  These  crystals  can 
also  be  obtained  from  crushed  insects,  watery  extracts  of 
various  internal  organs,  and  certain  other  substances, 
so  that  they  are  not  absolute  proof  of  the  presence  of 
semen.  Negative  results,  upon  the  other  hand,  are  con- 
clusive, even  when  the  semen  is  many  years  old. 


DIAGNOSIS    OF   RABIES  45 1 

The  reagent  consists  of  iodin,  2.54  gm.;  potassium 
iodid,  1.65  gm.;  and  distilled  water,  30  c.c. 

DIAGNOSIS  OF  RABIES 

In  view  of  the  brilliant  results  attending  prophylactic 
treatment  by  the  Pasteur  method,  early  diagnosis  of 
rabies  (hydrophobia)  in  animals  which  have  bitten  per- 
sons is  extremely  important. 

The  most  reliable  means  of  diagnosis  is  the  production 
of  the  disease  in  a  rabbit  by  subdural  or  intracerebral 
injection  of  a  little  of  the  filtrate  from  an  emulsion  of 
the  brain  and  medulla  of  the  suspected  animal.  The 
diagnosis  can,  however,  usually  be  quickly  and  easily 
made  by  microscopic  demonstration  of  Negri  bodies. 
Whether  these  bodies  be  protozoan  in  nature  and  the 
cause  of  the  disease,  as  is  held  by  many,  or  whether  they 
be  products  of  the  disease,  it  is  certain  that  their  pres- 
ence is  pathognomonic. 

Negri  bodies  are  sharply  outlined,  round,  oval,  or 
somewhat  irregular  structures  which  vary  in  size,  the 
extremes  being  0.5  and  18  fi.  They  consist  of  a  hyalin- 
like  cytoplasm,  in  which  when  properly  stained  one  or 
more  chromatin  bodies  can  usually  be  seen.  They  are 
situated  chiefly  within  the  cytoplasm  of  the  large  cells 
of  the  central  nervous  system,  the  favorite  location 
being  the  multipolar  cells  of  the  hippocampus  major 
(Anmion's  horn).  In  many  cases  they  suggest  red  blood- 
corpuscles  lying  within  nerve-cells. 

Probably  the  best  clinical  method  of  demonstrating 
Negri  bodies  is  the  impression  method  of  Langdon 
Frothingham,  which  is  carried  out  as  described  on  page 

*v   L' L  l_  l_  l£  IZ    0I~     v.'c   I  L  »^I    .a]  I 


452  MISCELLANEOUS    EXAMINATIONS 

(i)  Place  the  dog's  brain^  upon  a  board  about  lo  inches 
square,  and  divide  into  two  halves  by  cutting  along  the  me- 
dian line  with  scissors. 

(2)  From  one  of  the  halves  cut  away  the  cerebellum  and 
open  the  lateral  ventricle,  exposing  the  Ammon's  horn. 

(3)  Dissect  out  the  Ammon's  horn  as  cleanly  as  possible. 

(4)  Cut  out  a  small  disk  at  right  angles  to  the  long  axis  of 
the  Ammon's  horn,  so  that  it  represents  a  cross-section  of  the 
organ. 

(5)  Place  this  disk  upon  the  board  near  the  edge,  with  one 
of  the  cut  surfaces  upward. 

(6)  Press  the  surface  of  a  thoroughly  clean  slide  upon  the 
disk  and  lift  it  suddenly.  The  disk  (if  its  exposed  surface  has 
not  been  allowed  to  become  too  dry)  will  cling  to  the  board, 
leaving  only  an  impression  upon  the  slide.  Make  several 
similar  impressions  upon  different  portions  of  the  slide,  using 
somewhat  greater  pressure  each  time.  Impressions  are  also 
to  be  made  from  the  cut  surface  of  the  cerebellum,  since  Negri 
bodies  are  sometimes  present  in  the  Purkinje  cells  when  not 
found  in  the  Ammon's  horn. 

(7)  Before  the  impressions  dry,  immerse  in  methyl-alcohol 
for  one-half  to  two  minutes. 

(8)  Cover  with  Van  Gieson's  methylene-blue-fuchsin  stain, 
warming  gently  for  one-half  to  two  minutes.  This  stain,  as 
modified  by  Frothingham,  is  as  follows.  It  must  be  freshly 
mixed  each  day: 

Tap-water 20  c.c; 

Saturated  alcoholic  solution  basic  fuchsin i  drop; 

("Fuchsin  f.  Bac,"  Griibler). 
Saturated  aqueous  solution  methylene-blue i  drop. 

("Methylenblau  f.  Bac,"  Grubler). 

(9)  Wash  in  water  and  dry  with  filter-paper.  Examine 
with  a  low  power  to  locate  the  large  cells  in  which  the  bodies 

1  For  Dr.  Frothingham's  method  of  removing  a  dog's  brain  see  Ameri- 
can Journal  of  Public  Hygiene  for  February,  1908. 


PLATE   Xlll 


A' 


•-'■■£>'''■•. 


^f^' 


■w- 


Nerve-cells  containing  Negri  bodies. 

Hippocampus  impression  preparation,  dog.  Van  Gieson  stain; 
X  looo.  T,  Negri  bodies;  2,  capillary;  3,  free  red  blood-corpuscles 
(courtesy  of  Langdon  Frothingham). 


DIAGNOSIS   OF  RABIES  453 

are  apt  to  be  found,  and  study  these  with  an  oil-immersion 
lens. 

The  Negri  bodies  are  stained  a  pale  pink  to  purplish  red, 
and  frequently  contain  small  blue  dots  (Plate  XIII).  The 
nerve-cells  are  blue,  and  red  blood-corpuscles  are  colorless 
or  yellowish-copper  colored. 

When  the  work  is  finished,  the  board  with  the  dissected 
brain  is  sterilized  in  the  steam  sterilizer. 

Demonstration  of  Negri  bodies  by  this  method  is  quicker 
and,  probably,  more  certain  than  by  the  study  of  sections. 
It  has  the  decided  advantage  over  the  smear  method  that  the 
histologic  structure  is  retained.  One  or  more  of  the  impres- 
sions generally  shows  the  entire  cell  arrangement  almost  as 
well  as  in  sections,  and  it  is  very  easy  to  locate  favorable 
fields  with  a  i6-mm.  objective. 


CHAPTER  Vm 
BACTERIOLOGIC  METHODS 

Bacteriology  has  become  so  important  a  part  of 
medicine  that  some  knowledge  of  bacteriologic  methods 
is  imperative  for  the  present-day  practitioner.  It  has 
been  the  plan  of  this  book  to  describe  the  various  bacteria 
and  bacteriologic  methods  with  the  subjects  to  which 
they  seemed  to  be  particularly  related.  The  tubercle 
bacillus  and  its  detection,  for  example,  are  described  in 
the  chapters  upon  Sputum  and  Urine;  blood-cultures 
are  discussed  in  the  chapter  upon  Blood,  There  are, 
however,  certain  methods,  notably  the  preparation  of 
media  and  the  study  of  bacteria  by  cultures,  which  do 
not  come  within  the  scope  of  any  previous  section,  and 
an  outline  of  these  is  given  in  the  present  chapter. 

L  APPARATUS 

Much  of  the  apparatus  of  the  clinical  laboratory  is 
called  into  use.    Only  the  following  need  special  mention : 

I.  Sterilizers. — Two  are  required. 

The  dry,  or  hot-air  sterilizer,  is  a  double-walled  oven 
similar  to  the  detached  ovens  used  with  gas  and  gasolene 
stoves.  It  has  a  hole  in  the  top  for  a  perforated  cork 
with  thermometer. 

The  steam  sterilizer  is  preferably  of  the  Arnold  type, 
opening  either  at  the  top  or  the  side.    Ah  autoclave,  which 

454 


APPARATUS  455 

sterilizes  with  steam  under  pressure,  is  very  desirable, 
but  not  necessary.  An  aluminum  pressure  cooker  is  a 
very  satisfactory  substitute  for  the  autoclave.  It  costs 
about  fifteen  dollars. 

2.  Incubator. — This  is  the  most  expensive  piece  of 
apparatus  which  will  be  needed.  It  is  made  of  copper, 
and  has  usually  both  a  water-  and  an  air-jacket  sur- 
rounding the  incubating  chamber.  It  is  provided  with 
thermometer,  thermo-regulator,  and  some  source  of  heat, 
usually  a  Koch  safety  Bunsen  burner.  With  a  little 
ingenuity  one  can  rig  up  a  drawer  or  a  small  box,  in 
which  a  fairly  constant  temperature  can  be  maintained 
by  means  of  an  electric  light.  The  degree  of  heat  can 
be  regulated  by  moving  the  drawer  in  or  out,  or  holes 
can  be  made  in  which  corks  may  be  inserted  and  re- 
moved as  needed.  A  Thermos  bottle  has  been  suggested 
as  a  temporary  make-shift. 

3 .  Culture-tubes  and  Flasks. — For  most  work  ordinary 
test-tubes,  5  by  f  inches,  are  satisfactory.  For  special 
purposes  a  few  3  by  §  inch  and  6  by  f  inch  tubes  may 
be  needed.  Heavy  tubes,  which  do  not  easily  break,  can 
be  obtained,  and  are  especially  desirable  when  tubes  are 
cleaned  by  an  untrained  assistant.  The  tubes  are  usually 
stored  in  wire  baskets. 

Flasks  of  various  sizes  are  needed.  The  Ehrlenmeyer 
type  is  best.  Quart  and  pint  milk  bottles  and  2 -ounce 
wide-mouthed  bottles  will  answer  for  most  purposes. 

4.  Platinum  Wires. — At  least  two  of  these  are  needed. 
Each  consists  of  a  piece  of  platinum  wire  about  8  cm. 
long,  fixed  in  the  end  of  a  glass  or  metal  rod.  One  is 
made  of  about  22  gage  wire  and  its  end  is  curled  into  a 
loop  I  to  2  mm.  in  diameter.    A  loop  i  mm.  in  diameter  is 


456  BACTERIOLOGIC  METHODS 

sometimes  called  a  "normal."    The  other  wire  is  some- 
what heavier  and  its  tip  is  hammered  flat. 

5.  Pipets,  etc. — In  addition  to  the  graduated  pipets 
with  which  every  laboratory  is  supplied,  there  are  a 
number  of  forms  which  are  generally  made  from  glass 
tubing  as  needed.  One  of  the  simplest  of  these  is  made 
as  follows:  A  section  of  glass  tubing,  about  12  cm.  long 
and  5  mm.  in  diameter,  is  grasped  at  the  ends,  and  its 


6r?OUP>  A 


Gf?OUP    B 


Fig.  169. — Process  of  making  pipets  (Group  A)  and  Wright's  capsule  (Group  B).    The 
dotted  lines  indicate  where  the  glass  is  to  be  broken. 

center  is  heated  in  a  concentrated  flame.  A  blast-lamp 
is  best,  but  a  Bunsen  burner  will  usually  answer,  par- 
ticularly if  fitted  with  a  "wing"  or  "fish-tail"  attachment. 
When  the  glass  is  thoroughly  softened  it  is  removed 
from  the  flame,  and,  with  a  steady,  but  not  rapid  pull, 
is  drawn  out  as  shown  in  Fig.  169.  The  slender  portion 
is  scratched  near  the  middle  with  a  file  and  is  broken  to 
make  two  pipets,  which  are  then  fitted  with  rubber 
nipples.     Two  conditions  are  essential  to  success:  the 


STERILIZATION  457 

glass  must  be  thoroughly  softened  and  it  must  be  re- 
moved from  the  flame  before  beginning  to  pull. 

A  nipple  can  be  made  of  a  short  piece  of  rubber 
tubing,  one  end  of  which  is  plugged  with  a  glass  bead. 

This  pipet  has  many  uses  about  the  laboratory.  When 
first  made  it  is  sterile  and  may  be  used  to  transfer  cul- 
tures. With  a  grease-pencil  mark  about  2  cm.  from  its 
tip  (see  Fig.  172),  it  is  useful  for  measuring  very  small 
quantities  of  fluid,  as  in  making  dilutions  for  the  Widal 
test  and  in  counting  bacteria  in  vaccines.  Mett's  tubes 
for  pepsin  estimation  may  be  made  from  the  capillary 
portion.  The  capillary  portion  also  makes  a  very  satis- 
factory blood-lancet  if  the  center  is  heated  in  a  low  flame 
and  the  two  ends  pulled  quickly  apart. 

Another  useful  device  is  the  Wright  capusle,  which 
is  made  as  shown  in  Fig.  169.  Its  use  is  illustrated  in 
Fig.  173.  After  the  straight  end  is  sealed  the  curved 
portion  may  be  hooked  over  the  aluminum  tube  of  the 
centrifuge,  and  the  contained  blood  or  other  fluid  sedi- 
mented ;  but  the  speed  should  not  be  so  great  as  to  break 
the  capsule. 

n.  STERILIZATION 

All  apparatus  and  materials  used  in  bacteriologic 
work  must  be  sterilized  before  use. 

Glassware,  metal,  etc.,  are  heated  in  the  hot-air  steril- 
izer at  150°  to  180°  C.  for  half  an  hour.  Flasks,  bottles, 
and  tubes  are  plugged  with  cotton  before  heating. 
Petri  dishes  may  be  wrapped  in  paper  in  sets  of  three. 
Pipets  and  glass  and  metal  hypodermic  syringes  are 
placed  in  cotton-stoppered  test-tubes. 

Culture-media  and  other  fluids  must  be  sterilized  by 
steam.    Exposure  in  an  autoclave  to  a  temperature  of 


4S8  BACTERIOLOGIC  METHODS 

iio°  C.  (6  pounds'  pressure)  for  one-half  hour  is  sufficient. 
With  the  Arnold  sterilizer,  which  is  more  commonly 
used,  the  intermittent  plan  must  be  adopted,  since 
steam  at  ordinary  pressure  will  not  kill  spores.  This  con- 
sists in  steaming  for  thirty  to  forty-five  minutes  on  three 
or  four  successive  days.  Spores  which  are  not  destroyed 
upon  the  first  day  develop  into  the  vegetative  form  and 
are  destroyed  at  the  next  heating.  Gelatin  media  must 
not  be  exposed  to  steam  for  more  than  twenty  minutes 
at  a  time,  and  must  then  be  removed  from  the  sterilizer 
and  cooled  in  cold  water,  otherwise  the  gelatin  may  lose 
its  power  to  solidify. 

Cotton  and  gauze  are  sterilized  by  either  hot  air  or 
steam,  preferably  the  latter. 

m,  PREPARATION  OF  CULTURE-TUBES 

New  tubes  should  be  washed  in  a  very  dilute  solution  of 
nitric  acid,  rinsed  in  clear  water,  and  allowed  to  drain  dry. 

Tubes  which  contain  dried  culture-media  are  cleaned 
with  a  test-tube  brush  after  boiling  in  a  strong  solution  of 
washing-soda.  They  are  then  rinsed  successively  in 
clear  water,  acidulated  water,  and  clear  water,  and  al- 
lowed to  drain. 

The  tubes  are  now  ready  to  be  plugged  with  raw  cotton 
— the  "cotton  batting"  of  the  dry  goods  stores.  This  is 
done  by  pushing  a  wad  of  cotton  into  each  tube  to  a 
depth  of  about  3  cm.  with  a  glass  rod.  The  plugs  should 
fit  snugly,  but  not  too  tightly,  and  should  project  from 
the  tube  sufficiently  to  be  readily  grasped  by  the  fingers. 
The  tubes  are  next  placed  in  wire  baskets  and  heated  in 
an  oven  for  about  one-half  hour  at  150°  C.  in  order  to 
mold  the  stoppers  to  the  shape  of  the  tubes.    The  heat- 


CULTURE-MEDIA  459 

ing  should  not  char  the  cotton,  although  a  slight  brown- 
ing does  no  harm.  The  tubes  are  now  ready  to  be  filled 
with  culture-media. 

IV.  CULTURE-MEDIA 

For  a  careful  study  of  bacteria  a  great  variety  of  cul- 
ture-media is  required,  but  only  a  few — bouillon,  agar  or 
solidified  blood-serum,  and  gelatin — are  much  used  in 
routine  work.  A  great  deal  of  work  can  be  done  with  a 
single  medium,  for  which  purpose  solidified  blood- 
serum  is  probably  best.  The  ordinary  culture-media, 
put  up  in  tubes  ready  for  use,  can  be  purchased  through 
any  pharmacy. 

Preparation  of  Culture-media. — 

Beef  Infusion 

Hamburger  steak,  lean 500  gm.; 

Tap-water 1000  c.c. 

Mix  well;  let  soak  about  twenty-four  hours  in  an  ice- 
chest,  and  squeeze  through  cheese-cloth.  This  infusion 
is  not  used  by  itself,  but  forms  the  basis  for  various 
media.  "Double  strength"  infusion,  used  in  making 
agar-agar,  requires  equal  parts  of  the  meat  and  water. 

Infusion  Bouillon 

Beef  infusion 1000  c.c; 

Peptone  (Witte) 10  gm. ; 

Salt S    " 

Boil  until  dissolved;  bring  to  original  bulk  with  water; 
adjust  reaction,  and  filter. 

Beef  Extract  Bouillon 

Liebig's  extract  of  beef 3  gm.; 

Peptone 10    " 

Salt S    " 

Tap-water locx)  c.c. 


460  BACTERIOLOGIC  METHODS 

Heat  until  all  ingredients  are  dissolved,  cool,  and  beat 
in  the  whites  of  two  eggs;  bring  slowly  to  the  boiling- 
point  again;  boil  briskly  for  five  minutes  and  filter.  It 
is  not  usually  necessary  to  adjust  the  reaction. 

Agar-agar 
Preparation  of  this  medium  usually  gives  the  student 
much  trouble.    There  should  be  no  difficulty  if  the  direc- 
tions are  carefully  carried  out. 

Agar-agar,  powdered  or  in  shreds 15  gm.; 

Tap- water 500  c.c. 

Boil  until  thoroughly  dissolved  and  add — 

Peptone 10  gm.; 

Salt S    " 

When  these  have  dissolved,  replace  the  water  lost  in 
boiling,  cool  to  about  60°  C,  and  add  500  c.c.  double- 
strength  beef  infusion.  Bring  slowly  to  the  boil,  adjust- 
ing the  reaction  meanwhile,  and  boil  for  at  least  five 
minutes.  Filter  while  hot  through  a  moderately  thick 
layer  of  absorbent  cotton  wet  with  hot  water  in  a  hot 
funnel.  A  piece  of  coarse  wire  gauze  should  be  placed 
in  the  funnel  underneath  the  cotton  to  give  a  larger  filter- 
ing surface.  This  medium  will  be  clear  enough  for  or- 
dinary work.  If  an  especially  clear  agar  is  desired,  it 
can  be  filtered  through  paper  in  an  Arnold  sterilizer. 

Agar  can  also  be  made  by  boiling  15  gm.  of  powdered 
agar  in  1000  c.c.  of  bouillon  until  dissolved,  replacing 
the  water  lost  in  boiling,  and  filtering  through  paper 
in  a  sterilizer.    It  can  be  cleared  with  egg  if  desired. 

Glycerin  Agar-agar 
To  1000  c.c.  melted  agar  add  60  to  70  c.c.  glycerin. 


CULTURE-MEDIA  46 1 

Gelatin 

Dissolve  100  to  120  gm.  "golden  seal"  gelatin  in  1000 
c.c.  nutrient  bouillon  with  as  little  heat  as  possible, 
adjust  the  reaction,  cool,  beat  in  the  whites  of  two  eggs, 
bring  slowly  to  the  boiling  point,  boil  for  a  few  minutes, 
and  filter  hot  through  filter-paper  wet  with  hot  water. 
Sterilize  in  an  Arnold  sterilizer  for  twenty  minutes  upon 
three  successive  days  and  cool  in  cold  water  after  each 
heating. 

Sugar  Media 

Any  desired  sugar  may  be  added  to  bouillon,  agar,  or 
gelatin  in  proportion  of  10  gm.  to  the  liter.  Dextrose 
is  most  frequently  required.  When  other  sugars  are 
added,  media  made  from  beef-extract  should  be  used, 
since  those  made  from  beef-infusion  contain  enough 
dextrose  to  cause  confusion. 

Loffler's  Blood-serum 

Dextrose-bouillon  (i  per  cent.) i  part; 

Blood-serum 3  parts. 

Mix  and  tube.  Place  in  an  inspissator  at  the  proper 
slant  for  three  to  six  hours  at  80°  to  90°  C.  When  firmly 
coagulated,  sterilize  in  the  usual  way.  A  large  "double- 
cooker"  makes  a  satisfactory  inspissator.  The  tubes 
are  placed  in  the  inner  compartment  at  the  proper  slant, 
a  lid  with  perforation  for  a  thermometer  is  applied,  and 
the  whole  is  weighted  down  in  the  water  of  the  outer 
compartment. 

Blood-serum  is  obtained  as  follows:  Beef  or  pig  blood 
is  collected  in  a  bucket  at  the  slaughter-house  and 
placed  in  an  ice-chest  until  coagulated.  The  clot  is  then 
gently  loosened  from  the  wall  of  the  vessel.    After  about 


462  BACTERIOLOGIC  METHODS 

twenty-four  hours  the  serum  will  have  separated  nicely 
and  can  be  siphoned  off.  It  is  then  stored  in  bottles 
with  a  little  chloroform  until  needed.  Red  cells,  if 
abundant,  darken  the  medium,  but  do  no  harm. 

Solidified  blood-serum  is  probably  the  most  satisfac- 
tory medium  for  general  purposes.  Nearly  all  patho- 
genic organisms  grow  well  upon  it. 

Hemoglobin  Medium 

The  simplest  way  to  prepare  this  is  to  smear  a  drop 
of  blood,  obtained  by  puncture  of  the  finger,  over  the 
surface  of  an  agar-slant,  and  to  incubate  over  night  to 
make  sure  of  sterility.  It  is  used  chiefly  for  growing  the 
influenza  bacillus.  It  may  be  noted  that  the  bacillus 
will  not  grow  well  on  blood  from  a  person  who  has 
recently  recovered  from  influenza. 

Litmus  Milk 

Fresh  milk  is  steamed  in  an  Arnold  sterilizer  for  half 
an  hour,  and  placed  in  the  ice-chest  over  night.  The 
milk  is  siphoned  off  from  beneath  the  cream,  and  suffi- 
cient aqueous  solution  of  Htmus  or,  preferably,  azolitmin 
is  added  to  give  a  blue-violet  color.  It  is  then  tubed  and 
sterilized. 

Potato 

CyUnders  about  |  inch  thick  are  cut  from  po- 
tato and  split  obliquely.  These  wedge-shaped  pieces 
are  soaked  over  night  in  running  water  and  placed, 
broad  ends  down,  in  large  tubes,  in  the  bottom  of  which 
is  placed  a  little  cotton  saturated  with  water.  They  are 
sterilized  for  somewhat  longer  periods  than  ordinary 
media. 


CULTURE-MEDIA  463 

Dunham's  Peptone  Soltjtion 

Peptone 10  gm.; 

Salt 5    " 

Water 1000  c.c. 

Dissolve  by  boiling;  filter,  tube,  and  sterilize. 

This  medium  is  used  to  determine  indol  production. 
To  a  twenty-four-  to  forty-eight-hour-old  culture  is 
added  5  to  lo  drops  of  concentrated  chemically  pure 
sulphuric  acid  and  i  c.c.  of  i  :  10,000  solution  of  sodium 
nitrite.  Appearance  of  a  pink  color  shows  the  presence 
of  indol.  A  pink  color  before  the  nitrite  is  added  shows 
the  presence  of  both  indol  and  nitrites. 

Hiss'  Serum  Media 

Blood-seriim i  part; 

Water 3  parts. 

Warm  and  adjust  reaction  to  -I-0.2  to  4-0.8.  Add 
litmus  or  azolitmin  solution  to  give  a  blue-violet  color. 
Finally,  add  i  per  cent,  of  inulin  or  any  desired  sugar. 
The  inulin  medium  is  very  useful  in  distinguishing  be- 
tween the  pneumococcus  and  streptococcus. 

Bile  Medium 

Ox-  or  pig-bile  is  obtained  at  the  slaughter-house, 
tubed,  and  sterilized.  This  is  used  especially  for  grow- 
ing typhoid  bacilli  from  the  blood  during  life.  The  fol- 
lowing is  probably  as  satisfactory  as  fresh  bile  and  is 
more  convenient: 

Inspissated  ox-bile  (Merck) 30.0  gm.; 

Peptone 2.5    " 

Water 250.0  c.c. 

Dissolve,  place  in  tubes,  and  sterilize. 


464  BACTERIOLOGIC  METHODS 

Reaction  of  Media. — The  chemical  reaction  of  the 
medium  exerts  a  marked  influence  upon  the  growth  of 
bacteria.  It  is  adjusted  after  all  ingredients  are  dis- 
solved by  adding  sufficient  caustic  soda  solution  to 
overcome  the  acidity  of  the  meat  and  other  substances 
used.  In  general,  the  most  favorable  reaction  lies  be- 
tween the  neutral  points  of  Utmus  and  phenolphtha- 
lein,  representing  a  very  faint  alkaUnity  to  litmus. 
In  routine  work  it  is  usually  sufficient  to  test  with 
litmus-paper.  When  greater  accuracy  is  demanded, 
the  following  method  should  be  used:  After  all  ingre- 
dients are  dissolved  and  the  loss  during  boiling  has 
been  replaced  with  water,  10  c.c.  of  the  medium  are 
transferred  to  an  evaporating  dish,  diluted  with  40  c.c. 
of  water,  and  boiled  for  three  minutes  to  drive  off  carbon 
dioxid.  One  c.c.  of  0.5  per  cent.  alcohoUc  solution  of 
phenolphthalein  is  then  added,  and  decinormal  sodium 
hydroxid  solution  is  run  in  from  a  buret  until  the  neutral 
point  is  reached,  indicated  by  the  appearance  of  a  per- 
manent pink  color.  The  number  of  cubic  centimeters  of 
decinormal  solution  required  to  bring  this  color  indicates 
the  number  of  cubic  centimeters  of  normal  sodium  hy- 
droxid solution  which  will  be  required  to  neutralize  100 
c.c.  of  the  medium.  The  standard  reaction  is  +1.5, 
which  means  that  the  medium  must  be  of  such  degree 
of  acidity  that  1.5  c.c.  of  normal  solution  would  be  re- 
quired to  neutralize  100  c.c.  This  corresponds  to  faint 
alkalinity  to  litmus.  Most  pathogenic  bacteria  grow 
better  with  a  reaction  of  -|-  i.o  or  -fo.S.  Example:  If  the 
10  c.c.  which  were  titrated  required  2  c.c.  of  decinormal 
solution  to  bring  the  pink  color,  the  reaction  is  -|-  2 ;  and 
0.5  c.c.  of  normal  sodium  hydroxid  must  be  added  to 


STAINING  METHODS  465 

each  100  c.c.  of  the  medium  to  reduce  it  to  the  standard, 

+  1.5- 

Tubing  Culture-media. — The  finished  product  is 
stored  in  flasks  or  distributed  into  test-tubes.  This  is 
done  by  means  of  a  funnel  fitted  with  a  section  of  rubber 
tubing  with  a  glass  tip  and  a  pinch-cock.  Great  care 
must  be  exercised,  particularly  with  media  which  sohd- 
ify,  not  to  smear  any  of  them  upon  the  inside  of  the  mouth 
of  the  tube,  otherwise  the  cotton  stopper  will  stick.  Tubes 
are  generally  filled  to  a  depth  of  3  or  4  cm.  For  stab- 
cultures  a  greater  depth  is  required. 

After  tubing,  all  culture-media  must  be  sterilized  as 
already  described.  Agar-tubes  are  cooled  in  a  slanting 
position  to  secure  the  proper  surface  for  inoculation. 

Storage  of  Cultiure-media.^ — All  media  should  be 
stored  in  a  cool  place,  preferably  an  ice-chest.  Evapora- 
tion may  be  prevented  by  covering  the  tops  of  the  tubes 
with  tin-foil  or  the  rubber  caps  which  are  sold  for  the 
purpose;  or  the  cotton  stopper  may  be  pushed  in  a  short 
distance  and  a  cork  inserted. 

V.  STAINING  METHODS 

In  general,  bacteria  are  stained  to  determine  their 
morphology,  their  reaction  with  special  methods  {e.  g., 
Gram's  method),  and  the  presence  or  absence  of  certain 
structures,  as  spores,  flagella,  and  capsules.  Staining 
methods  for  various  purposes  and  the  formulae  of  the 
staining  fluids  have  been  given  in  previous  chapters  and 
can  be  found  by  consulting  the  Index.  The  following 
will  probably  be  most  frequently  used: 

Methods  for  tubercle  bacilli  (see  pp.  64-66  and  205). 

Methods  for  capsules  of  bacteria  (see  pp.  72  and  421). 

30 


466  BACTERIOLOGIC  METHODS 

Methods  for  Treponema  pallidum  (see  p.  447). 
Method  for  gonococcus  (see  p.  422). 
Loffler's  alkaline  methylene-blue  (see  p.  74). 
Blood-stains  (see  pp.  266-271). 

The  method  of  staining  for  morphology  is  as  follows, 

using  any  bacterial  stain: 

(i)  Make  a  thin  smear  upon  a  slide  or  cover-glass.  Heavy 
grease-pencil  marks  across  the  slide  will  limit  the  stain  to  any 
portion  desired. 

(2)  Dry  in  the  air,  or  by  warming  high  above  the  flame, 
where  one  can  comfortably  hold  the  hand. 

(3)  "Fix"  by  passing  the  preparation,  film  side  up,  rather 
slowly  through  the  flame  of  a  Bun  sen  burner:  a  cover-glass 
three  times,  a  slide  about  twelve  times.  If  the  film  takes  on 
a  brownish  discoloration,  most  marked  about  the  edges,  it 
has  been  scorched  and  is  worthless.  Smears  can  also  be 
fixed  by  flaming  with  alcohol,  as  described  for  blood-films 
(see  p.  265). 

(4)  Apply  the  stam  for  the  necessary  length  of  time,  gen- 
erally one-quarter  to  one  minute. 

(5)  Wash  in  water. 

(6)  Dry  by  waving  high  above  a  flame  or  by  blotting  with 
filter-paper. 

(7)  Mount  by  pressing  the  cover,  fihn  side  down,  upon  a 
drop  of  Canada  balsam  on  a  slide.  Slides  may  be  examined 
with  the  oil-immersion  lens  without  a  cover-glass. 

Simple  Bacterial  Stains. — A  simple  solution  of  any 
basic  anilin  dye  (methylene-blue,  basic  fuchsin,  gentian 
violet,  etc.)  will  stain  nearly  all  bacteria.  These  simple 
solutions  are  not  much  used  in  the  cUnical  laboratory, 
because  other  stains,  such  as  Loffler's  methylene-blue  and 


STAINING  METHODS  467 

Pappenheim's  pyronin-methyl-green  stain,  which  serve 
the  purpose  even  better,  are  at  hand. 

Pappenheim's  P3n"onin-methyl-green  Stain. — This  so- 
lution colors  bacteria  red  and  nuclei  of  cells  blue.  It 
is,  therefore,  especially  useful  for  intracellular  bacteria 
like  the  gonococcus  and  the  influenza  bacillus.  It  is  a 
good  stain  for  routine  purposes,  and  is  a  most  excellent 
contrast  stain  for  Gram's  method.  It  colors  the  cyto- 
plasm of  lymphocytes  bright  red,  and  has  been  used  as  a 
differential  stain  for  these  cells.  The  solution  is  applied 
cold  for  one-half  to  five  minutes.  It  consists  of  saturated 
aqueous  solution  methyl-green,  3  to  4  parts,  and  satu- 
rated aqueous  solution  pyronin,  i  to  i§  parts.  It  is  a 
good  plan  to  keep  these  solutions  in  stock  and  to  mix  a 
new  lot  of  the  staining  fluid  about  once  a  month.  If  it 
stains  too  deeply  with  either  dye,  the  proper  balance  is 
attained  by  adding  a  little  of  the  other. 

Carbol  Thionin. — Saturated  solution  thionin  in  50  per 
cent,  alcohol,  20  c.c. ;  2  per  cent,  aqueous  solution  phenol, 
100  c.c. 

This  stain  is  especially  useful  in  coimting  bacteria  for 
standardization  of  vaccines  (see  p.  483).  It  can  be  used 
as  a  general  stain.  In  blood  work  it  is  used  for  the  ma- 
larial parasite  and  for  demonstration  of  basophilic  degen- 
eration of  the  red  cells.  The  fluid  is  applied  for  one- 
half  to  three  minutes,  after  fixation  by  heat,  or  about 
a  minute  in  saturated  aqueous  solution  of  mercuric 
chlorid  or  i  per  cent,  formalin  in  alcohol. 

Gram's  Method. — This  is  a  very  useful  aid  in  differen- 
tiating certain  bacteria  and  should  be  frequently  re- 
sorted to.  It  depends  upon  the  fact  that  when  treated 
successively    with    gentian- violet    and    iodin,    certain 


468  BACTERIOLOGIC  METHODS 

bacteria  (owing  to  formation  of  insoluble  compounds) 
retain  the  stain  when  subsequently  treated  with  alco- 
hol, whereas  others  quickly  lose  it.  The  former  are 
called  Gram-positive;  the  latter,  Gram-negative.  In 
order  to  render  Gram-negative  organisms  visible,  some 
contrasting  counterstain  is  commonly  applied,  but  this 
is  not  a  part  of  Gram's  method  proper. 

(i)  Make  smears,  dry,  and  fix  by  heat. 

(2)  Apply  anilin-gentian-violet  or  formalin-gentian-violet 
(see  p.  74)  two  to  five  minutes. 

(3)  Wash  with  water. 

(4)  Apply  Gram's  iodin  solution  one-half  to  two  minutes. 

(5)  Wash  in  alcohol  until  the  purple  color  ceases  to  come 
off.  This  is  conveniently  done  in  a  watch-glass.  The  prep- 
aration is  placed  in  the  alcohol,  face  downward,  and  one 
edge  is  raised  and  lowered  with  a  needle.  As  long  as  any 
color  is  coming  off,  purple  streaks  will  be  seen  diffusing  into 
the  alcohol  where  the  surface  of  the  fluid  meets  the  smear. 
If  forceps  be  used,  beware  of  stain  which  may  have  dried 
upon  them.  The  thinner  portions  of  smears  from  pus  should 
be  practically  colorless  at  this  stage.  If  the  smears  resist 
decolorization  the  gentian- violet  and  iodin  solution  should 
be  applied  for  a  shorter  time,  say,  one-half  minute  each. 

(6)  Apply  a  contrast  stain  for  one-half  to  one  minute. 
The  stains  commonly  used  for  this  purpose  are  an  aqueous 
or  alcoholic  solution  of  Bismarck  brown  and  a  weak  solution 
of  fuchsin.  In  the  writer's  experience,  Pappenheim's  pyro- 
nin-methyl -green  mixture  is  much  more  satisfactory;  it 
brings  out  Gram-negative  bacteria  more  sharply,  and  is 
especially  desirable  for  intracellular  Gram-negative  organ- 
isms like  the  gonococcus  and  influenza  bacillus,  since  the 
bacteria  are  bright  red  and  nuclei  of  cells  blue. 

(7)  Wash  in  water,  dry,  and  mount  in  balsam. 


STAINING  METHODS  469 

The  more  important  bacteria  react  to  this  staining 
method  as  follows: 

Graii  Staining  Gram  Decolorizing 

(Deep  purple).  (Colorless  unless  a  counterstain  is  used). 

Staphylococcus.  Gonococcus. 

Streptococcus.  Meningococcus. 

Pneumococcus.  Micrococcus  catarrhalis. 

Bacillus  diphtheriae.  Bacillus  of  influenza. 

Bacillus  tuberculosis.  Tj^ihoid  bacillus. 

Bacillus  of  anthrax.  Bacillus  coli  communis. 

Bacillus  of  tetanus.  Spirillum  of  Asiatic  cholera. 

Bacillus  aerogenes  capsulatus.  Bacillus  pyocyaneus. 

Bacillus  of  Friedlander. 

Koch- Weeks  bacillus. 

Bacillus  of  Morax-Axenfeld. 

Moller's  Method  for  Spores. — Bodies  of  bacteria  are 
blue,  spores  are  red. 

(i)  Make  thin  smears,  dry,  and  fix, 

(2)  Wash  in  chloroform  for  two  minutes. 

(3)  Wash  in  water. 

(4)  Apply  5  per  cent,  solution  of  chromic  acid  one-half  to 
two  minutes. 

(5)  Wash  in  water. 

(6)  Apply  carbolfuchsin  and  heat  to  boiling. 

(7)  Decolorize  in  5  per  cent,  solution  of  sulphuric  acid. 

(8)  Wash  in  water. 

(9)  Apply  I  per  cent,  aqueous  solution  of  methylene-blue 
one-half  minute. 

(10)  Wash  in  water,  dry,  and  mount. 

Huntoon's  Method  for  Spores. — This  is  a  new  method 
which  is  simple  and  appears  to  be  very  reliable.  Spores 
are  deep  red,  bodies  of  bacteria  are  blue. 


47©  BACTERIOLOGIC  METHODS 

(i)  Make  a  rather  thick  smear,  dry,  and  fix  in  the  usual 
way. 

(2)  Apply  as  much  of  the  stain  as  will  remain  on  the 
cover-glass,  and  steam  over  a  flame  for  one  minute,  replacing 
the  stain  lost  by  evaporation. 

(3)  Wash  in  water.    The  film  is  bright  red. 

(4)  Dip  the  preparation  a  few  times  into  a  weak  solution 
of  sodium  carbonate  (7  or  8  drops  of  saturated  solution  in  a 
glass  of  water).  Too  long  application  of  the  carbonate  will 
cause  the  spores  to  be  blue. 

(5)  The  instant  the  fihn  turns  blue,  rinse  well  in  water. 

(6)  Dry,  moimt,  and  examine. 
Preparation  of  Stain. — 

(i)  Acid  fuchsin  (Grubler) 4  gm.; 

Aqueous  solution  acetic  acid  (2  per  cent.) ...  .50  c.c. 

(2)  Methylene-blue  (Grubler) 2  gm.; 

Aqueous  solution  acetic  acid  (2  per  cent.) 50  c.c. 

Mix  the  two  solutions,  let  stand  for  fifteen  minutes,  and 
filter  off  the  voluminous  precipitate  through  moistened 
filter-paper.  The  filtrate  is  the  staining  fluid.  It  keeps 
several  weeks,  but  requires  filtration  when  a  precipitate 
forms. 

Loffler's  Method  for  Flagella. — The  methods  for 
flagella  are  applicable  only  to  cultures.  Enough  of  the 
growth  from  an  agar-culture  (which  should  not  be  more 
than  eighteen  to  twenty-four  hours  old)  to  produce  faint 
cloudiness  is  added  to  distilled  water.  A  small  drop  of 
this  is  placed  on  a  cover-glass,  spread  by  tilting,  and  dried 
quickly.  The  covers  must  be  absolutely  free  from  grease. 
To  insure  this,  they  may  be  warmed  in  concentrated 
sulphuric  acid,  washed  in  water,  and  kept  in  a  mixture 


METHODS    OF   STUDYING   BACTERIA  47 1 

of  alcohol  and  strong  ammonia.    When  used  they  are 
dried  upon  a  fat-free  cloth. 

(i)  Fix  by  heating  the  cover  over  a  flame  while  holding 
in  the  fingers. 

(2)  Cover  with  freshly  filtered  mordant  and  gently  warm 
for  about  a  minute. 

The  mordant  consists  of: 

Aqueous  solution  of  tannic  acid  (20  per  cent.) lo  c.c; 

Saturated  solution  ferrous  sulphate,  cold S    " 

Satiuated  aqueous  or  alcoholic  solution  gentian- violet. .   i     " 

(3)  Wash  in  water. 

(4)  Apply  freshly  filtered  anilin-gentian-violet,  warming 
gently  for  one-half  to  one  minute. 

(5)  Wash  in  water,  dry,  and  moimt  in  balsam. 

VL  METHODS  OF  STUDYING  BACTERIA 

The  purpose  of  bacteriologic  examinations  is  to  de- 
termine whether  bacteria  are  present  or  not,  and,  if 
present,  their  species  and  comparative  numbers.  In 
general,  this  is  accomplished  by:  i,  Direct  microscopic 
examination;  2,  Cultural  methods;  3,  Animal  inocula- 
tion. 

1.  Direct  Microscopic  Examination. — Every  bac- 
teriologic examination  should  begin  with  a  microscopic 
study  of  smears  from  the  pathologic  material,  stained 
with  a  general  stain,  by  Gram's  method,  and  often  by 
the  method  for  the  tubercle  bacillus.  This  yields  a 
great  deal  of  information  to  the  experienced  worker, 
and  in  many  cases  is  all  that  is  necessary  for  the  pur- 
pose in  view.  It  will  at  least  give  a  general  idea  of  what 
is  to  be  expected,  and  may  determine  future  procedure. 


472  BACTERIOLOGIC  METHODS 

2.  Cultural  Methods.— (i)  Collection  of  Material.— 

Material  for  examination  must  be  collected  under  abso- 
lutely aseptic  conditions.  It  may  be  obtained  with  a 
platinum  wire — which  has  been  heated  to  redness  just 
previously  and  allowed  to  cool — or  with  a  swab  of  sterile 
cotton  on  a  stiff  wire  or  wooden  applicator.  Such  swabs 
may  be  placed  in  cotton-stoppered  test-tubes,  sterilized, 
and  kept  on  hand  ready  for  use.  Fluids  which  contain 
very  few  bacteria,  and  hence  require  that  a  considerable 
quantity  be  used,  may  be  collected  in  a  sterile  hypo- 
dermic syringe  or  one  of  the  pipets  described  on  p.  456. 
The  method  of  obtaining  blood  for  cultures  is  given 
on  p.  293. 

(2)  Inoculating  Media. — The  material  is  thoroughly 
distributed  over  the  surface  of  some  solid  medium,  solid- 
ified blood-serum  being  probably  the  best  for  routine 
work.  When  previous  examination  of  smears  has  shown 
that  many  bacteria  are  to  be  expected,  a  second  tube 
should  be  inoculated  from  the  first,  and  a  third  from  the 
second,  so  as  to  obtain  isolated  colonies  in  at  least  one 
of  the  tubes.  The  platinum  wire  must  be  heated  to 
redness  before  and  ajter  each  inoculation.  When  only 
a  few  organisms  of  a  single  species  are  expected,  as  is  the 
case  in  blood-cultures,  a  considerable  quantity  of  the 
material  is  mixed  with  a  fluid  medium. 

(3)  Incubation. — Cultures  are  placed  in  an  incubator 
which  maintains  a  uniform  temperature,  usually  of  37.5° 
C,  for  eighteen  to  twenty-four  hours,  and  the  growth,  if 
any,  is  studied  as  described  later.  Gelatin  will  melt  with 
this  degree  of  heat,  and  must  be  incubated  at  about 
room-temperature. 

(4)  Study  of  Cultures. — When   the  original  culture 


METHODS    OF   STUDYING   BACTERIA  473 

contains  more  than  one  species,  they  must  be  separated, 
or  obtained  in  "pure  culture,"  before  they  can  be  studied 
satisfactorily.  To  accomplish  this  it  is  necessary  to  so 
distribute  them  on  solid  media  that  they  form  separate 
colonies,  and  to  inoculate  fresh  tubes  from  the  individual 
colonies.  In  routine  work  the  organisms  can  be  suffi- 
ciently distributed  by  drawing  the  infected  wire  over  the 
surface  of  the  medium  in  a  series  of  streaks.  If  a  suffi- 
cient number  of  streaks  be  made,  some  of  them  are 
sure  to  show  isolated  colonies.  Another  method  of 
obtaining  isolated  colonies  is  to  inoculate  the  water 
of  condensation  of  a  series  of  tubes,  the  first  from  the 
second,  the  second  from  the  third,  etc.,  and,  by  tilting, 
to  flow  the  water  once  over  the  surface  of  the  medium. 
One  or  more  of  these  tubes  will  be  almost  sure  to  show 
nicely  separated  colonies. 

In  order  to  determine  the  species  to  which  an  organism 
belongs  it  is  necessary  to  consider  some  or  all  of  the  fol- 
lowing points : 

(i)  Naked-eye  and  microscopic  appearance  of  the  col- 
onies on  various  media. 

(2)  Comparative  luxuriance  of  growth  upon  various 
media.  The  influenza  bacillus,  for  example,  can  be 
grown  upon  media  containing  hemoglobin,  but  not  on 
the  ordinary  media. 

(3)  Morphology,  special  staining  reactions,  and  the 
presence  or  absence  of  spores,  flagella,  and  capsules. 
Staining  methods  for  these  purposes  have  been  given. 

(4)  Motility.  This  is  determined  by  observing  the 
living  organism  with  an  oil-immersion  lens  in  a  hanging- 
drop  preparation,  made  as  follows:  A  small  drop  of  a 
bouillon  culture  or  of  water  of  condensation  from  an 


474  BACTERIOLOGIC  METHODS 

agar  or  blood-serum  tube  is  placed  upon  the  center  of 
a  cover-glass;  and  over  this  is  pressed  the  concavity  of 
a  "hollow-ground  slide"  previously  ringed  with  vaselin. 
The  slide  is  then  turned  over  so  as  to  bring  the  cover- 
glass  on  top.  In  focusing,  the  edge  of  the  drop  should 
be  brought  into  the  field.  Great  care  must  be  exer- 
cised not  to  break  the  cover  by  pushing  the  objective 
against  it. 

It  is  not  always  easy  to  determine  whether  an  organ- 
ism is  or  is  not  motile,  since  the  motion  of  currents  and 
the  Brownian  motion  which  affects  all  particles  in  sus- 
pension are  sometimes  very  deceptive. 

(5)  Production  of  chemical  changes  in  the  media. 
Among  these  are  coagulation  of  milk;  production  of  acid 
in  milk  and  various  sugar  media  to  which  li  mus  has  been 
added  to  detect  the  change;  production  of  gas  in  sugar 
media,  the  bacteria  being  grown  in  fermentation  tubes 
similar  to  those  used  for  sugar  tests  in  urine;  and  pro- 
duction of  indol. 

(6)  Ability  to  grow  without  oxygen.  For  anaerobic 
methods,  the  reader  is  referred  to  larger  works. 

(7)  Effects  produced  when  inoculated  into  animals. 
3.  Animal  Inoculation.— In    clinical   work    this   is 

resorted  to  chiefly  to  detect  the  tubercle  bacillus.  The 
method  is  described  on  p.  432. 

For  the  study  of  bacteria  in  cultures,  a  small  amount 
of  a  pure  culture  is  injected  subcutaneously  or  into  the 
peritoneal  ca\'ity.  The  animals  most  used  are  guinea- 
pigs,  rabbits,  and  mice.  For  intravenous  injection  the 
rabbit  is  used  because  of  the  easily  accessible  marginal 
vein  of  the  ear. 


CHARACTERISTICS    OF   SPECIAL   BACTERIA  475 

VIL  CHARACTERISTICS  OF  SPECIAL  BACTERIA 

Owing  to  the  great  number  of  bacterial  species,  most 
of  which  have  not  been  adequately  studied,  positive 
identification  of  an  unknown  organism  is  often  a  very 
difficult  problem.  Fortunately,  however,  only  a  few 
are  commonly  encountered  in  routine  work,  and  identi- 
fication of  these  with  comparative  certainty  presents  no 
great  difficulty.  Their  more  distinctive  characteristics 
are  outlined  in  this  section. 

1.  Staphylococcus  pyogenes  aureus. — ^The  morphology 
and  staining  reactions  (described  on  p.  420)  and  the  ap- 
pearance of  the  colonies  are  sufficient  for  diagnosis. 
Colonies  on  solidified  blood-serum  and  agar  are  roxmded, 
sUghtly  elevated,  smooth  and  shining,  and  vary  in  color 
from  light  yellow  to  deep  orange.  Young  colonies  are 
sometimes  white. 

2.  Staphylococcus  pyogenes  albus. — This  is  similar 
to  the  above,  but  colonies  are  white.  It  is  generally 
less  virulent. 

3.  Staphylococcus  pyogenes  citreus. — The  colonies  are 
lemon  yellow;  otherwise  it  resembles  the  white  staphylo- 
coccus. 

4.  Streptococcus  pyogenes. — The  morphology  and 
staining  reactions  have  been  described  (see  p.  421).  The 
chains  are  best  seen  in  the  water  of  condensation  and  in 
bouillon  cultures.  The  cocci  are  not  motile.  Colonies 
on  blood-serum  are  minute,  round,  grayish,  and  trans- 
lucent. Litmus  milk  is  usually  acidified  and  coagulated, 
although  slowly.  The  streptococcus  rarely  produces 
acid  in  Hiss'  serum-water-litmus-inulin  medium  (see 
p.  463). 


476  BACTERIOLOGIC  METHODS 

5.  Pneumococcus. — The  only  organism  with  which 
this  is  hkely  to  be  confused  is  the  streptococcus.  The 
distinction  is  often  extremely  difficult. 

Detection  of  the  pneumococcus  in  fresh  material  has 
been  described  (see  p.  71).  In  cultures  it  frequently 
forms  long  chains.  Capsules  are  not  present  in  cultures 
except  upon  special  media.  They  show  best  upon  a 
serum  medium  like  that  described  for  the  gonococcus, 
but  can  frequently  be  seen  in  milk.  Colonies  on 
blood-serum  resemble  those  of  the  streptococcus.  The 
pneumococcus  usually  promptly  acidifies  and  coagu- 
lates milk  and  acidifies  and  coagulates  Hiss'  serum- 
water  with  inulin.  The  latter  property  is  very 'helpful 
in  diagnosis. 

6.  Gonococcus. — Its  morphology  and  staining  pecu- 
Harities  are  given  on  p.  422.  These  usually  suffice  for 
its  identification,  cultural  methods  being  rarely  under- 
taken. In  cultures  the  chief  diagnostic  point  is  its 
failure  to  grow  on  ordinary  media.  To  grow  it  the 
most  convenient  medium  is  made  by  adding  ascitic  or 
hydrocele  fluid  (which  has  been  obtained  under  aseptic 
conditions)  to  melted  agar  in  proportion  of  i  part  of 
serum  to  3  parts  of  agar.  The  agar  in  tubes  is  melted 
and  cooled  to  about  45°  C;  the  serum  is  added  with  a 
pipet  and  mixed  by  shaking;  and  the  tubes  are  again 
cooled  in  a  slanting  position.  Colonies  upon  this 
medium  are  minute,  grayish,  and  translucent. 

7.  Diplococcus  intracellularis  meningitidis. — It  grows 
poorly  or  not  at  all  on  plain  agar.  On  Loffler's  blood- 
serum,  upon  which  it  grows  fairly  well,  colonies  are 
round,  colorless  or  hazy,  flat,  shining,  and  viscid  looking. 
It  quickly  dies  out. 


CHARACTERISTICS    OF   SPEQAL   BACTERIA  477 

8.  Diphtheria  Bacillus. — The  diagnosis  is  usually 
made  from  a  study  of  stained  smears  from  cultures 
upon  blood-senmi.  Its  morphology  and  staining  pecu- 
Uarities  are  characteristic  when  grown  on  this  medium 
(see  p,  435).  The  bacilli  are  non-motile  and  Gram- 
positive.  The  colonies  are  round,  elevated,  smooth, 
and  grayish. 

9.  Typhoid  and  Colon  Bacilli. — These  are  medium- 
sized,  motile.  Gram-negative,  non-spore-bearing  bacilli. 
Upon  blood-serum  they  form  rounded,  grayish,  sUghtly 
elevated,  viscid  looking  colonies,  those  of  the  colon  bacil- 
lus being  somewhat  the  larger.  They  do  not  liquefy 
gelatin.  They  represent  the  extremes  of  a  large  group 
with  many  intermediate  types.  They  are  distinguished 
as  follows: 

Typhoid  Bacillus.  Colon  Bacillus. 

Actively  motile.  Much  less  active. 

Growth  on  potato  usually  invisible.  Growth  distinctly  visible  as  dirty 

gray  or  brownish  slimy  layer. 

No  gas  produced  in  glucose  media.  Produces  gas. 

Growth  in  litmus  milk  produces  no  Litmus  milk  pink  and  coagulated. 

change. 

Produces  no  indol   in  Dunham's  Produces  indol.       (For   test,   see 

peptone  medium.  p.  463.) 

Agglutinates  with  senun  from  ty-  Does  not  agglutinate  with  typhoid 

phoid-fever  patient.     (Recently         serum. 

isolated  bacilli  do  not  agglutinate 

well.) 

ID.  Bacillus  of  Influenza. — Diagnosis  will  usually  rest 
upon  the  morphology  and  staining  peculiarities,  de- 
scribed on  p.  75,  and  upon  the  fact  that  the  bacillus  will 
not  grow  on  ordinary  media,  but  does  grow  upon  hemo- 
globin-containing media.     It  can  be  grown  upon  agar- 


478  BACTERIOLOGIC   METHODS 

slants  which  have  been  smeared  with  a  drop  of  blood 
from  a  pmicture  in  the  finger.  Before  inoculation  these 
slants  should  be  incubated  to  make  sure  of  sterility. 
The  colonies  are  difficult  to  see  without  a  hand  lens. 
They  are  very  minute,  discrete,  and  transparent,  re- 
sembling small  drops  of  dew. 

II.  Bacillus  of  Tuberculosis. — The  methods  of  iden- 
tifying this  important  organism  have  been  given  (see 
pp.  63,  205).  Cultivation  is  not  resorted  to  in  clinical 
work.  It  grows  very  slowly  and  only  on  certain  media. 
It  is  Gram-positive  and  non-motile. 


CHAPTER  IX 

PREPARATION  AND  USE  OF  VACCINES 

Bacterial  vaccines,  sometimes  called  "bacterins," 
which  within  the  past  few  years  have  come  to  play  an 
important  r61e  in  therapeutics,  are  suspensions  of  defi- 
nite numbers  of  dead  bacteria  in  normal  salt  solution. 
While  in  many  cases,  notably  in  gonorrhea  and  tuber- 
culosis, ready  prepared  or  "stock"  vaccines  are  satis- 
factory, it  is  usually  desirable  and  often  imperative  for 
best  results  to  use  vaccines  which  are  especially  prepared 
for  each  patient  from  bacteria  which  have  been  freshly 
isolated  from  his  own  lesion.  These  latter  are  called 
"autogenous  vaccines."  Only  through  them  can  one 
be  certain  of  getting  the  exact  strain  of  bacterium  which 
is  producing  the  disease. 

I.  PREPARATION  OF  VACCINE 

The  method  of  preparing  autogenous  vaccines  which 
is  used  in  the  author's  laboratory  is  here  described. 

1.  Preparation  of  Materials. — ^A  number  of  2-ounce 
wide-mouthed  bottles  are  cleaned  and  sterilized.  Each 
is  charged  with  50  c.c.  freshly  filtered  normal  salt  solu- 
tion (0.85  per  cent,  sodium  chlorid  in  distilled  water), 
and  is  capped  with  a  new  rubber  nursing-nipple,  without 
holes,  inverted  as  shown  in  Fig.  170.  A  small  section  of 
hollow  wire  or  a  hypodermic  needle  is  thrust  through  the 
cap  near  the  edge  to  serve  as  an  air  vent,  and  the  bottle 

479 


48o 


PREPARATION    AND   USE   OF   VACCINES 


and  contents  are  sterilized  in  an  autoclave.  If  an  auto- 
clave is  not  at  hand,  successive  steamings  in  an  Arnold 
sterilizer  will  answer,  provided  it  is  not  opened  between 
steamings.  After  sterilization,  the  pieces  of  wire  are 
pulled  out  and  the  holes  sealed  with  collodion. 

Most  workers  use  a  smaller  bottle  with  less  salt  solu- 
tion and  with  a  cotton  stopper;  and,  after  the  solution 


m  ••  luiflin"  '^""' 


Fig.  170. — Vaccine  bottles:  A,  Cap  ready  to  be  applied;  B,  ready  for  sterilization;  C, 
finished  vaccine. 


BACTERIAL  VACS 


Kc 


j)l-MJ? 


has  been  sterilized,  apply  a  specially  made  "vaccine  bot- 
tle cap."  The  method  which  has  been  detailed  above 
calls  for  an  unnecessarily  large  quantity  of  fluid  (which 
is  no  real  objection),  but  has  certain  advantages:  the 
nursing  nipples  are  easily  obtained  at  any  pharmacy; 
the  rubber  is  not  put  upon  the  stretch  as  is  the  case  with 
some  caps,  and  is,  therefore,  self -sealing;  no  cotton-lint 


PREPARATION   OF   VACCINE  48 1 

-falls  into  the  salt  solution  before  the  cap  is  applied;  and 
the  cap  offers  a  concavity  which  may  be  filled  with 
alcohol  for  sterilizing  before  the  needle  is  plunged 
through. 

A  number  of  test-tubes,  each  charged  with  10  c.c.  of 
normal  salt  solution  and  plugged  with  cotton,  are  also 
prepared  and  sterilized. 

2.  Obtaining  the  Bacteria.— A  culture  on  some  solid 
medium  is  made  from  the  patient's  lesion,  and  a  pure 
culture  is  obtained  in  the  usual  way.  This  preliminary 
work  should  be  carried  through  as  quickly  as  possible. 
K  for  any  reason  there  is  much  delay,  it  is  best  to  begin 
over  again,  the  experience  gained  in  the  first  trial  en- 
abhng  one  to  carry  the  second  through  more  rapidly. 
When  a  pure  culture  is  obtained,  a  number  of  tubes  of 
blood-serum  or  agar — lo  or  12  in  the  case  of  streptococ- 
cus or  pneumococcus,  4  or  5  in  the  case  of  most  other 
organisms — are  planted  and  incubated  over  night  or 
until  a  good  growth  is  obtained. 

3.  Making  the  Suspension. — A  few  cubic  centimeters 
of  the  salt  solution  from  one  of  the  lo-c.c.  salt-tubes  is 
transferred  by  means  of  a  sterile  pipet  to  each  of  the 
culture-tubes,  and  the  growth  thoroughly  rubbed  up 
with  a  stiff  platinum  wire  or  a  glass  rod  whose  tip  is 
bent  at  right  angles.  The  suspension  from  the  different 
tubes,  usually  amounting  to  about  10  c.c,  is  then  col- 
lected in  one  large  tube  (size  about  6  by  f  inches) ;  and 
the  upper  part  of  the  tube  is  drawn  out  in  the  flame  of  a 
blast  lamp  or  Bunsen  burner,  as  indicated  in  Fig.  171,  -B, 
a-short  section  of  glass  tubing  being  fused  to  the  rim  of  the 
tube  to  serve  as  a  handle.  It  is  then  stood  aside,  and 
when  cool  the  narrow  portion  is  sealed  off. 

31 


482  PREPARATION   AND   USE    OF   VACCINES 

The  resulting  hermetically  sealed  capsule  is  next 
thoroughly  shaken  for  ten  to  twenty  minutes  to  break 
up  all  clumps  of  bacteria.  Some  small  pieces  of  glass  or 
a  little  clean  sterile  sand  may  be  introduced  to  assist  in 
this,  but  with  many  organisms  it  is  not  necessary. 

4.  Sterilization. — ^The  capsule  is  placed  in  a  water-bath 
at  60°  C.  for  forty-five  minutes.    This  can  be  done  in 


Fig.  171. — Process  of  making  hermetically  sealed  capsules  containing  liquid. 

an  ordinary  rice-cooker,  with  double  lid,  through  which 
a  thermometer  is  inserted.  When  both  compartments 
are  filled  with  water  it  is  an  easy  matter  to  maintain  a 
uniform  temperature  by  occasional  application  of  a 
small  flame.  The  time  and  temperature  are  important: 
too  little  heat  will  fail  to  kill  the  bacteria,  and  too  much 
will  destroy  the  efficiency  of  the  vaccine. 
When  sterilization  is  complete  the  capsule  is  opened, 


PREPARATION    OF   VACCINE 


483 


a  few  drops  are  planted  on  agar  or 
blood-serum,  and  the  capsule  is  again 
sealed. 

5.  Counting. — When  incubation  of 
theplant  has  shown  the  suspension  to 
be  sterile  it  is  ready  for  counting. 

There  must  be  ready  a  number  of 
clean  slides;  a  few  drops  of  normal 
salt  solution  on  a  slide  or  in  a  watch- 
glass;  a  blood-lancet,  which  can  be 
improvised  from  a  spicule  of  glass  or 
a  pen;  and  two  capillary  pipets  with 
squarely  broken  off  tips  and  grease- 
pencil  marks  about  2  cm.  from  the 
tip  (Fig.  172).  These  are  easily  made 
by  drawing  out  a  piece  of  glass  tub- 
ing, as  described  on  page  456. 

It  is  necessary  to  work  quickly. 
After  thorough  shaking,  the  capsule 
is  opened  and  a  few  drops  forced  out 
upon  a  slide.  Any  remaining  clumps 
of  bacteria  are  broken  up  with  one 
of  the  pipets  by  holding  it  against  and 
at  right  angles  to  the  slide,  and  alter- 
nately sucking  the  fluid  in  and  forc- 
ing it  out.  The  pipet  is  most  easily 
controlled  if  held  in  the  whole  hand 
with  the  rubber  bulb  between  the 
thumb  and  the  side  of  the  index-finger. 
A  finger  is  then  pricked  until  a  drop 
of  blood  appears;  and  into  the  second 
pipet  are  quickly  drawn  successively: 


3    3 

s-  <» 


3     3 


484  PREPARATION   AND   USE    OF   VACaNES 

I  or  2  volumes  normal  salt  solution  (or,  better,  a  i  per 
cent,  solution  of  sodium  citrate  which  prevents  coagula- 
tion); a  small  bubble  of  air;  i  volume  of  blood;  a  small 
bubble  of  air;  and,  finally,  i  volume  of  bacterial  sus- 
pension. (A  'Volume"  is  measured  by  the  distance  from 
the  tip  of  the  pipet  to  the  grease-pencil  mark.)  The 
contents  of  the  pipet  are  then  forced  out  upon  a  slide 
and  thoroughly  mixed  by  sucking  in  and  out,  care  being 
taken  to  avoid  bubbles;  after  which  the  fluid  is  distributed 
to  a  number  of  slides  and  spread  as  in  making  blood- 
smears. 

The  films  are  stained  with  Wright's  blood-stain  or, 
better,  by  a  few  minutes'  application  of  carbol-thionin, 
after  fixing  for  a  minute  in  saturated  mercuric  chlorid 
solution.  With  an  oil-immersion  lens  both  the  red  cells 
and  the  bacteria  in  a  number  of  microscopic  fields  are 
counted.  The  exact  number  is  not  important;  for  con- 
venience 500  red  cells  may  be  counted.  From  the  ratio 
between  the  number  of  bacteria  and  of  red  cells,  it  is 
easy  to  calculate  the  number  of  bacteria  in  i  c.c.  of 
the  suspension,  it  being  known  that  there  are  5000  mil- 
lion red  corpuscles  in  a  cubic  centimeter  of  normal  human 
blood.  If  there  were  twice  as  many  bacteria  as  red  cor- 
puscles in  the  fields  counted,  the  suspension  would  con- 
tain 10,000  million  bacteria  per  cubic  centimeter. 

The  count  can  also  be  made  with  the  hemacytometer, 

using   a   weak   carbolfuchsin  or  gentian-violet,  freshly 

filtered,    as    diluting   fluid.      Callison    recommends    the 

following  diluting  fluid: 

Hydrochloric  acid 2  c.c; 

Mercuric  chlorid  (0.2  per  cent,  solution) 100    " 

Acid  fuchsin  (i  per  cent,  aqueous  solution),  to  color. 
The  color  should  be  just  deep  enough  not  to  obscure  the  ruled  lines. 


METHOD  OF  USE  485 

A  very  thin  cover-glass  must  be  used;  and,  after 
filling,  the  counting-chamber  must  be  set  aside  for  an 
hour  or  more  to  allow  the  bacteria  to  settle.  Mallory 
and  Wright  advise  the  use  of  the  shallow  chamber  manu- 
factured by  Zeiss  for  counting  blood-plates,  but  many  2- 
mm.  oil-immersion  objectives  have  sufl&cient  working  dis- 
tance to  allow  the  use  of  the  regular  counting-chamber, 
provided  a  very  thin  cover  is  used. 

6.  Diluting. — The  amount  of  the  suspension  which, 
when  diluted  to  50  c.c,  will  give  the  strength  desired  for 
the  finished  vaccine  having  been  determined,  this  amount 
of  salt  solution  is  withdrawn  with  a  hypodermic  syringe 
from  one  of  the  bottles  already  prepared,  and  is  replaced 
with  an  equal  amount  of  suspension.  One-tenth  c.c.  of 
trikresol  or  lysol  is  finally  added  and  the  vaccine  is  ready 
for  use.  To  prevent  possible  leakage  about  the  cap, 
the  neck  of  the  bottle  is  dipped  in  melted  paraffin.  The 
usual  strengths  are:  staphylococcus,  1000  million  in 
I  c.c;  most  other  bacteria,  100  million  in  i  c.c. 

n.  METHOD  OF  USE 

Vaccines  are  administered  subcutaneously,  usually  in 
the  arm  or  abdominal  wall  or  between  the  shoulder- 
blades.  The  technic  is  the  same  as  for  an  ordinary 
hypodermic  injection.  The  syringe  is  usually  sterilized 
by  boiling.  The  site  of  the  injection  may  be  mopped  with 
alcohol  or  may  be  touched  with  a  pledget  of  cotton 
saturated  with  tincture  of  iodin  or  liquor  cresolis  com- 
positus.  The  rubber  cap  of  the  container  is  sterilized 
J3y  filling  the  concavity  with  alcohol  for  some  minutes, 
usually  while  the  syringe  is  being  sterilized,  or  simply 
placing  a  drop  of  hquor  cresolis  compositus  upon  it. 


486  PREPARATION   AND   USE    OF   VACCINES 

The  bottle  is  then  inverted  and  well  shaken,  when  the 
needle  is  plunged  through  the  rubber  and  the  desired 
quantity  withdrawn.  The  hole  seals  itself.  The  most 
satisfactory  syringe  is  the  comparatively  inexpensive 
"Sub-Q  Tuberculin"  syringe  graduated  in  hundredths 
of  a  cubic  centimeter, 

EL  DOSAGE 

Owing  to  variations,  both  in  virulence  of  organisms 
and  susceptibility  of  patients,  no  definite  dosage  can  be 
assigned.  Each  case  is  a  separate  problem.  Wright's 
original  proposal  was  to  regulate  the  size  and  frequency 
of  dose  by  its  effect  upon  the  opsonic  index,  but  this  is 
beyond  the  reach  of  the  practitioner.  The  more  widely 
used  * 'clinical  method"  consists  in  beginning  with  a 
very  small  dose  and  cautiously  increasing  until  the 
patient  shows  either  improvement  or  some  sign  of  a 
"reaction,"  indicated  by  headache,  malaise,  fever,  ex- 
acerbation of  local  disease,  or  inflammatory  reaction  at 
the  site  of  injection.  The  reaction  indicates  that  the 
dose  has  been  too  large.  The  beginning  dose  of  staphy- 
lococcus is  about  50  milHon;  the  maximum,  1000  million 
or  more.  Of  most  other  organisms  the  beginning  dose 
is  5  milHon  to  10  million;  maximum,  about  100  million. 
Ordinarily,  injections  are  given  once  or  twice  a  week; 
very  small  doses  may  be  given  every  other  day. 

IV.  THERAPEUTIC  INDICATIONS 

The  therapeutic  effect  of  vaccines  depends  upon  their 
power  to  produce  active  immunity:  they  stimulate  the 
production  of  opsonins  and  other  antibacterial  sub- 
stances which  enable  the  body  to  combat  the  infecting 


THERAPEUTIC  INDICATIONS  487 

bacteria.  Their  especial  field  is  the  treatment  of  sub- 
acute and  chronic  localized  infections,  in  some  of  which 
they  offer  the  most  effective  means  of  treatment  at  our 
command.  In  most  chronic  infections  the  circulation  of 
blood  and  lymph  through  the  diseased  area  is  very 
sluggish,  so  that  the  antibodies,  when  formed,  cannot 
readily  reach  the  seat  of  disease.  Ordinary  measures 
which  favor  circulation  in  the  diseased  part  should, 
therefore,  accompany  the  vaccine  treatment.  Among 
these  may  be  mentioned  incisions  and  drainage  of  ab- 
scesses, dry  cupping,  application  of  heat.  Bier's  hyper- 
emia, etc.,  but  such  measures  should  not  be  applied 
during  the  twenty-four  hours  succeeding  an  injection, 
since  the  first  effect  of  the  vaccine  may  be  a  temporary 
lowering  of  resistance.  Vaccines  are  of  little  value,  and, 
in  general,  are  contraindicated  in  very  acute  infections, 
particularly  in  those  which  are  accompanied  by  much 
systemic  intoxication,  for  in  such  cases  the  power  of  the 
tissues  to  produce  antibodies  is  already  taxed  to  the 
limit.  It  is  true,  nevertheless,  that  remarkably  bene- 
ficial results  have  occasionally  followed  their  use  in  such 
acute  conditions  as  malignant  endocarditis,  but  here 
they  should  be  tried  with  extreme  caution. 

Probably  best  results  are  obtained  in  staphylococcus 
infections,  although  pneumococcus,  streptococcus,  and 
colon  bacillus  infections  usually  respond  nicely.  Among 
clinic.al  conditions  which  have  been  treated  successfully 
with  vaccines  are  furunculosis,  acne  vulgaris,  infected 
operation-wounds,  pyelitis,  cystitis,  subacute  otitis 
media,  osteomyelitis,  infections  of  nasal  accessory  si- 
huses,  etc.  Vaccine  treatment  of  the  mixed  infection  is 
doubtless   an   important   aid   in   tuberculosis   therapy, 


488  PREPARATION    AND   USE   OF   VACaNES 

and  in  some  cases  the  result  is  brilliant.  When,  as  is 
common,  several  organisms  are  present  in  the  sputum, 
a  vaccine  is  made  from  each,  excepting  the  tubercle 
bacillus,  of  which  autogeneous  vaccines  are  not  used  in 
practice.  To  avoid  the  delay  and  consequent  loss  of 
virulence  entailed  by  study  and  isolation  of  the  several 
varieties,  many  workers  make  the  bacterial  suspension 
directly  from  the  primary  cultures.  The  resulting  vac- 
cines contain  all  strains  which  are  present  in  the  sputum 
in  approximately  the  same  relative  numbers.  Although 
open  to  criticism  from  a  scientific  standpoint,  this 
method  offers  decided  practical  advantages  in  many 
cases. 

It  has  been  shown  that  vaccines  are  useful  in  prevent- 
ing as  well  as  curing  infections.  Their  value  has  been 
especially  demonstrated  in  typhoid  fever.  Three  or  four 
doses  of  about  lOo  million,  200  million,  400  million,  and 
600  million  typhoid  bacilli  are  given  about  five  to  seven 
days  apart.  Results  in  the  army,  where  the  plan  has 
been  tried  on  a  large  scale,  show  that  such  vaccination 
is  effective. 

V.  TUBERCULINS 

Tuberculins  contain  the  products  of  tubercle  bacilli 
or  their  ground-up  bodies,  the  latter  class  being  prac- 
tically vaccines.  They  are  undoubtedly  of  great  value 
in  the  treatment  of  localized  tuberculosis,  particularly 
of  bones,  joints,  and  glands;  and  are  of  rather  indefinite 
though  certainly  real  value  in  chronic  pulmonary  tuber- 
culosis, especially  when  the  disease  is  quiescent.  The 
best  known  are  Koch's  old  tuberculin  (T.  O.),  bouillon 
filtrate  (B.  F.),  triturate  residue  (T.  R.),  and  bacillary 
emulsion  (B.  E.).    There  seems  to  be  Uttle  difference  in 


TUBERCULINS  489 

the  actions  of  these,  although  theoretically  T.  R.  should 
immunize  against  the  bacillus  and  B.  F.  against  its 
toxic  products.  The  choice  of  tuberculin  is  much  less 
important  than  the  method  of  administration.  The 
making  of  autogenous  tuberculins  is  impracticable, 
hence  stock  preparations  are  used  in  practice. 

Since  the  dose  is  exceedingly  minute,  the  tuberculin 
as  purchased  must  be  greatly  diluted  before  it  is  avail- 
able for  use.  A  convenient  plan  is  to  use  the  rubber- 
capped  bottles  of  sterile  salt  solution  described  for  vac- 
cines (see  p.  479),  adding  sufficient  tuberculin  to  give 
the  desired  strength,  together  with  o.i  c.c.  trikresol  to 
insure  sterihty.  The  practitioner  should  bear  in  mind 
that  while  tuberculin  is  capable  of  good,  it  is  also  capable 
of  great  harm.  Everything  depends  upon  the  dosage 
and  plan  of  treatment.  Probably  a  safe  beginning  dose 
for  a  pulmonary  case  is  o.ooooi  milligram  of  B.  E.,  B.  P., 
or  T.  R.;  for  gland  and  bone  cases,  about  o.oooi  milli- 
gram. O.  T.  is  now  used  chiefly  in  diagnosis.  The 
intervals  are  about  one  week  or,  rarely,  three  days,  when 
very  small  doses  are  given.  The  dose  is  increased 
steadily,  but  with  extreme  caution;  and  should  be  dimin- 
ished or  temporarily  omitted  at  the  first  indication  of  a 
"reaction,"  of  which,  in  general,  there  are  three  forms: 

{a)  General. — Elevation  of  temperature  (often  slight), 
headache,  malaise. 

(6)  Local. — Increase  of  local  symptoms,  amount  of 
sputum,  etc. 

(c)  Stick. — Inflammatory  reaction  at  site  of  injection. 

Treatment  is  usually  continued  until  a  maximum  dose 
of  I  miUigram  is  reached,  the  course  extending  over  a 
year  or  more. 


490  PREPARATION   AND   USE   OF   VACCINES 

VL  TUBERCULIN  IN  DIAGNOSIS 
The  tissues  of  a  tuberculous  person  are  sensitized 
toward  tuberculin,  and  a  reaction  (see  preceding  section) 
occurs  when  any  but  the  most  minute  quantity  of  tuber- 
culin is  introduced  into  the  body.  Non-tuberculous 
persons  exhibit  no  such  reaction.  This  is  utilized  in 
the  diagnosis  of  obscure  forms  of  tuberculosis,  the  test 
being  applied  in  a  number  of  ways: 

1.  Hypodermic  Injection. — Koch's  old  tuberculin  is 
used  in  successive  doses,  several  days  apart,  of  o.ooi ,  o.oi , 
and  O.I  mg.  A  negative  result  with  the  largest  amount 
is  considered  final.  The  reaction  is  manifested  by  fever 
within  eight  to  twenty  hours  after  the  injection.  The 
method  involves  some  danger  of  lighting  up  a  latent 
process,  and  has  been  largely  displaced  by  safer  methods. 

2.  Calmette's  Ophthalmo-reaction. — One  or  two  drops 
of  0.5  per  cent,  purified  old  tuberculin  are  instilled  into 
one  eye.  Tubercuhn  ready  prepared  for  this  purpose  is 
on  the  market.  If  tuberculosis  exists  anywhere  in  the 
body,  a  conjunctivitis  is  induced  within  twelve  to  twenty- 
four  hours.  This  generally  subsides  within  a  few  days. 
The  method  is  not  without  some,  though  slight,  risk  of 
injury  to  the  eye;  and  the  test  is  absolutely  contraindi- 
cated  in  the  presence  of  any  form  of  ocular  disease. 
A  second  instillation  should  not  be  tried  in  the  same  eye. 

3.  More  Reaction. — ^A  50  per  cent,  ointment  of  old 
tuberculin  in  lanolin  is  rubbed  into  the  skin  of  the  abdo- 
men, a  piece  about  the  size  of  a  pea  being  required. 
Dermatitis,  which  appears  in  twenty-four  to  forty-eight 
hours,  indicates  a  positive  reaction.  The  ointment  can 
be  purchased  ready  for  use. 

4.  Von  Pirquet's  Method. — This  is  the  most  satis- 


CUTANEOUS   TEST   FOR   SYPHIUS  49 1 

factory  of  the  tuberculin  tests.  Two  small  drops  of 
old  tuberculin  are  placed  on  the  skin  of  the  front  of  the 
forearm,  about  2"  inches  apart,  and  the  skin  is  slightly 
scarified,  first  at  a  point  midway  between  them,  and  then 
through  each  of  the  drops.  A  convenient  scarifier  is  a 
piece  of  heavy  platiniun  wire,  the  end  of  which  is  ham- 
mered to  a  chisel  edge.  A  wooden  tooth-pick  with  a 
chisel-shaped  end  is  also  convenient.  This  is  held  at 
right  angles  to  the  skin,  and  rotated  six  to  twelve  times 
with  just  sufficient  pressure  to  remove  the  epidermis 
without  drawing  blood.  In  about  ten  minutes  the 
excess  of  tuberculin  is  gently  wiped  away  with  cotton. 
No  bandage  is  necessary.  A  positive  reaction  is  shown 
by  the  appearance  in  twenty-four  to  forty-eight  hours 
of  a  papule  with  red  areola,  which  contrasts  markedly 
with  the  small  red  spot  left  by  the  control  scarification. 

These  tests  have  very  great  diagnostic  value  in  chil- 
dren, especially  those  under  three  years  of  age,  but  are 
often  misleading  in  adults,  positive  reactions  occurring 
in  many  apparently  healthy  individuals.  Negative 
tests  are  very  helpful  in  deciding  against  the  existence 
of  tuberculosis. 

Vn.  CUTANEOUS  TEST  FOR  SYPHILIS 

Noguchi  has  prepared  a  substance  called  luetin,  which 
produces  a  cutaneous  reaction  in  syphilis  similar  to  the 
tuberculin  skin  reaction  in  tuberculosis.  Luetin  con- 
sists of  ground  cultures  of  Treponema  pallidum  sterilized 
.  and  preserved  with  trikresol. 

A  small  drop  (0.05  c.c.)  of  luetin  is  injected  into  the 
skin  (not  under  it)  of  one  arm.    A  similar  preparation 


492  PREPARATION   AND   USE   OF   VACaNES 

without  the  treponema  is  injected  into  the  skin  of  the 
other  arm  as  a  control.  A  positive  reaction  usually 
begins  within  forty-eight  hours  and  consists  of  an  in- 
flammatory induration,  papule,  or  pustule.  It  is  some- 
times delayed  three  or  even  four  weeks. 

The  test  is  specific  for  syphilis.  It  is  present  in  late 
secondary,  tertiary,  latent,  and  hereditary  syphilis,  but 
is  usually  absent  in  primary  and  untreated  secondary 
cases.  In  general  paralysis  and  tabes  dorsaUs  it  is 
inconstant. 

Compared  with  the  Wassermann  reaction  it  is  more 
constant  in  tertiary  and  latent  syphilis,  while  the  Wasser- 
mann is  more  constant  in  primary  and  secondary  cases. 
Unlike  the  Wassermann,  the  reaction  does  not  disappear 
with  treatment,  but  persists  probably  until  a  complete 
cure  is  effected. 


CHAPTER  X 

SERODIAGNOSTIC  METHODS  ^ 
L  IMMUNITY 

With  two  exceptions  the  diagnostic  methods  here 
described  depend  on  one  or  another  law  of  immunity. 
These  laws  are  customarily  described  in  terms  of  Ehrlich's 
side-chain  theory.  It  is  not  practicable  to  undertake  a 
detailed  discussion  of  the  theory  here,  and  I  shall, 
accordingly,  confine  myself  to  such  discussion  and  de- 
finition of  the  bodies  concerned  as  will  enable  the  reader 
to  imdertake  the  reactions  himself  with  a  reasonably 
intelligent  conception  of  their  mechanism. 

Acquired  immunity,  that  form  of  immunity  resulting 
from  an  attack  of  a  given  disease,  depends  upon  the 
formation  within  the  body,  under  the  influence  of  the 
disease-producing  agent,  or  "antigen,"  of  bodies  possess- 
ing the  power  to  neutralize  the  poisons  produced  by  the 
antigen,  or  to  destroy  or  otherwise  affect  the  antigen 
itself.  Since  the  action  of  these  bodies  is  specific  {i.  e., 
they  act  only  on  the  particular  antigen  whose  presence 
has  led  to  their  production) ,  the  search  for  them  may  be 
resorted  to  for  diagnostic  purposes  whenever  they  can 
be  found  more  easily  than  can  the  antigen  itself.  With 
certain  exceptions,  to  be  noted  later,  the  presence  of  one 
or  other  of  these  bodies  may  be  regarded  as  pathogno- 
monic of  the  "corresponding  disease. 

1  By  Ross  C.  \Vhitman,  B.A.,  M.D.,  Professor  of  Surgical  Pathology 
and  Serology  in  the  University  of  Colorado. 

493 


494  SERODIAGNOSTIC   METHODS 

The  several  "immune  bodies"  act  by  means  of  different 
mechanisms,  by  virtue  of  which  they  may  be  classified 
in  three  groups — the  three  orders  of  receptors  of  Ehr- 
lich's  side-chain  theory.  With  the  first  group  we  are 
not  immediately  concerned. 

1.  Receptors  of  the  First  Order. — These  are  receptors 
which  serve  simply  as  connecting  links  between  the 
disease-producing  agent  (or,  rather,  of  its  toxin)  and  the 
tissues.  Under  the  influence  of  the  antigen  they  are 
produced  in  excess,  and  are  finally  set  free  in  the  circu- 
lation. Here  they  seize  upon  and,  so  to  speak,  satu- 
rate the  free  valence  of  the  antigen,  while  it  also  is  still 
free  in  the  blood  and  lymph,  in  such  a  way  as  to  leave 
the  latter  no  chemical  affinities  by  means  of  which  it 
may  combine  with  similar  bodies  still  in  relation  with 
the  cells.  The  antigen  is  thus  rendered  inert.  This 
order  of  receptors  includes  only  the  antitoxins;  for 
example,  those  of  diphtheria  and  tetanus. 

2.  Receptors  of  the  Second  Order. — These  have  a  com- 
bining group  similar  to  that  of  the  first  order,  and,  in 
addition,  a  group  possessing  a  ferment-Uke  action,  by 
means  of  which  the  characteristic  action  of  the  body  is 
effected.  The  ferment,  or  zymophore,  group  is  readily 
destroyed  by  heat,  so  that  serum  to  be  used  for  any  of 
the  purposes  included  in  the  group  must  not  be  heated. 
The  group  includes  the  agglutinins,  responsible  for  the 
several  appUcations  of  the  Widal  reaction;  the  precipi- 
tins, responsible  for  one  of  the  biologic  methods  to  be 
described  later  for  the  forensic  identification  of  blood- 
stains; and  the  opsonins. 

3.  Receptors  of  the  Third  Order. — These  bodies  con- 
sist of  two  combining  affinities  only,    One  of  these  com- 


APPARATUS  495 

bines  with  suitable  analogous  groups  of  the  antigen,  the 
other  combines  with  a  substance  which  is  called  com- 
plement because  it  "complements"  or  supplements  or 
completes  the  specific  action  of  the  immune  substance. 
Complement  is  normally  present  in  the  blood,  but  is 
unable  to  act  upon  the  antigen  without  the  mediation 
and  aid  of  the  immune  body.  The  latter  is,  therefore, 
called  the  amboceptor  or  'tween  body.  It  is  (relatively) 
thermostabile  and  keeps  practically  indefinitely  imder 
suitable  conditions.  It  is  to  be  remembered  that  this  is 
the  specific  immune  substance  whose  presence  or  ab- 
sence is  indicative  of  the  presence  or  absence  of  the  cor- 
responding disease.  The  native,  normally  present  com- 
plement is  (relatively)  thermolabile,  being  destroyed 
in  a  few  minutes  by  a  temperature  of  54°  to  56°  C, 
and  keeps  only  a  few  hours  under  the  best  conditions. 
It  is  non-specific,  and  within  certain  limits  the  comple- 
ment of  one  species  may  be  substituted  for  that  of  another. 
Thus,  in  the  Wassermann  reaction,  complement  contain- 
ing fresh  serum  from  guinea-pigs  is  usually  substituted 
for  the  normally  present  complement  of  the  patient's 
serum,  after  the  latter  has  been  destroyed. 

This  group  contains  the  lysins  and  the  bodies  respon- 
sible for  the  various  applications  of  the  complement- 
deviation  method  to  the  diagnosis  of  syphilis  (Was- 
sermann reaction),  gonorrhea,  cancer,  typhoid  fever, 
forensic  identification  of  blood,  etc. 

n.  APPARATUS 

-  Before  the  description  of  the  several  tests  is  taken  up, 
I  shall  give,  to  save  space,  a  list  of  general  equipment 
needed  for  such  work.     Special  apparatus  needed  for 


496 


SERODIAGNOSTIC   METHODS 


some  of  the  tests  will  be  mentioned  in  connection  with 
these. 

1.  Centrifuge. — While  the  usual  small  electric  or 
water-driven  instrument  can  be  employed,  a  larger 
machine,  capable  of  holding  4  or  8  tubes  of  about  50-c.c. 
capacity,  is  desirable. 

2.  Scales,  about  o.i  to  100  gm.  capacity. 

3.  Microscope,  magnifying  50  to  750  diameters. 

4.  Incubator  at  37°  C. 

5.  Water-bath,  to  be  regulated  as  required. 


c:)    c:>    c:^    <^ 

C3>     CP     c^    o     o 
CD     cs>     cr>     <:^     <:^ 
o      CZ)     cz>     <=C^^^ 

CD       <:Z>      CD        CD  \      p     CD 
CD       dD       CD>        CD       LJ      CD 


^ 


T5        <0     CD 
CD        CD       CD 
CD       CD      CD       CZ 
CD        C:D       CD        CD 
CD       CD       CD       CD 


^ 


Fig.  173. — Convenient  test-tube  rack  for  serum  work. 

6.  A  large  number  of  test-tubes,  about  §  x  5  inches, 

7.  Test-tube  racks  to  accommodate  the  above.  A 
double  row  of  holes  is  very  convenient.  Still  better  is  a 
special  rack  (Fig.  173),  made  of  copper  or  zinc,  with 
six  rows  of  holes,  six  to  each  row.  A  sheet  of  metal 
midway  between  top  and  bottom  contains  holes  to  cor- 
respond, so  that  tubes  are  held  without  danger  of  tip- 
ping. The  rack  holds  tubes  enough  for  18  Wassermann 
reactions,  if  but  one  antigen  is  used.  A  similar  rack, 
made  round,  and  with  the  two  lower  sheets  of  metal 


THE   WIDAL  REACTION  497 

small  enough  to  go  through  the  circular  opening  of  the 
water-bath,  while  the  top  sheet  is  larger,  so  as  to  rest 
on  the  edge  of  the  opening,  is  also  very  convenient. 

8.  Volumetric  pipets,  o.i  c.c.  in  one-one-hundredths, 
and  10  c.c.  in  one- tenths.  The  graduation  should  start 
near  the  point  where  the  emptying  of  the  pipet  is  stopped 
by  capillarity. 

9.  Capillary  pipets  (see  Fig.  172),  made  from  soft- glass 
tubing,  as  described  on  page  456.  The  tube  should  be 
of  such  a  size  that  the  ordinary  medicine-dropper  nipple 
will  fit  it  snugly.  Such  pipets  are  useful  for  a  variety 
of  purposes.  After  being  used  once  they  should  be 
thrown  away. 

10.  Glass  Capsules. — These  may  be  purchased  or, 
with  a  little  practice,  can  be  readily  prepared  from  the 
same  sort  of  tubing  by  drawing  out  a  piece  at  both  ends, 
and  sealing  in  the  flame.  If  desired,  one  end  may  be 
bent  over  to  form  a  hook  at  the  point  where  the  narrow- 
ing begins  (see  Fig.  169). 

11.  An  all-glass  syringe,  such  as  the  Luer,  about  5-c.c. 
capacity,  with  a  fairly  large  needle,  say  19  or  20  gage, 
preferably  of  platinum. 

m.  REACTIONS  BASED  UPON  IMMUNE  BODIES  OF  THE 
SECOND  ORDER 

A.  The  Widal  Reaction 

The  test  may  be  employed  for  the  diagnosis  of  a  variety 
of  infections,  e.  g.,  typhoid,  paratyphoid,  bacillary  dysen- 
tery, the  plague,  Asiatic  cholera,  epidemic  meningitis, 
etc.  In  clinical  work  it  is  used  only  for  the  diagnosis 
of  typhoid  and  paratyphoid  infections. 

32 


498 


SERODIAGNOSTIC   METHODS 


I.  Materials  Required. — The  following  especial  equip- 
ment is  needed: 

(i)  A  homogeneous  suspension  of  the  organism  or 
organisms  suspected  of  causing  the  disease.  Such  sus- 
pensions may  be  purchased  from  the  manufacturers  of 
biologic  preparations,  or  may  be  prepared  by  the  worker 
himself.  In  the  latter  case  twenty-four-hour-old  agar- 
slant  cultures  (preferably  attenuated  by  long-continued 
growth  on  culture-media)   should  be  washed  off  with 


Fig.  174. — Method  of  obtaining  blood  in  a  Wright  capsule:  A,  Filling  the  capsule;  B, 
the  bulb  has  been  warmed  and  the  capillary  end  sealed  in  the  flame;  C,  cooling  of  the 
capsule  has  drawn  the  blood  to  the  sealed  end;  D.  the  serum  has  separated,  and  the 
top  of  the  capsule  has  been  broken  off. 

normal  salt  solution  (0.85-0.9  per  cent,  sodium  chlorid), 
containing  either  0.5  per  cent,  phenol  or  o.i  per  cent, 
formalin,  and  shaken  until  the  suspension  is  as  uniform 
as  possible.  Dilute  by  adding  more  of  the  salt  solution 
until  the  suspension  is  but  slightly  milky,  and  preserve 
in  the  ice-box.  Such  a  suspension  will  keep  for  months. 
Shake  thoroughly  before  using.  The  suspension  will 
settle  less  rapidly  if  10  per  cent,  lactose  is  added  to  it. 
Suspensions  which  show  any  tendency  to  spontaneous 
agglutination  cannot,  of  course,  be  used. 


THE   WIDAL  REACTION  499 

(2)  Instead  of  the  suspension  of  killed  bacteria,  living 
young  cultures  (not  over  twenty-four  hours  old)  of  at- 
tenuated organisms  can  be  employed. 

(3)  About  0.1  c.c.  of  the  patient's  serum.  This  may  be 
obtained  by  pricking  the  cleansed  finger  or  ear  rather 
deeply  and  collecting  the  blood  in  one  of  the  capsules 
above  mentioned,  as  is  indicated  in  Fig.  174.  More 
than  one  capsule  should  be  at  hand,  so  that  a  fresh  one 
may  be  substituted  if  the  first  is  plugged  by  fibrin  before 
enough  blood  is  obtained. 

When  the  capsules  are  not  at  hand,  blood  may  be  ob- 
tained in  little  vials  such  as  may  be  made  by  breaking  off  the 
lower  f  inch  of  the  tubes  which  have  contained  peptoniz- 
ing powder.  Vials  in  which  hypodermic  tablets  are  sold  can 
be  used,  but  are  somewhat  too  narrow.  They  must,  of  course, 
be  well  cleaned.  One  of  these  is  filled  to  a  depth  of  about  I 
inch  from  a  puncture  in  the  ear,  and  is  then  set  aside  for  a 
few  hours.  When  the  clot  has  separated  it  is  picked  out  with 
a  needle,  leaving  the  serum. 

Sufficient  blood  may  also  be  collected  by  allowing 
drops  to  dry  on  glass  or  unglazed  paper  (without  heat- 
ing), to  be  afterward  macerated  in  water.  In  this  case, 
however,  dilutions  can  only  be  made  approximately. 

(4)  Glides,  preferably  hollow  ground,  cover-glasses, 
vaselin. 

2.  Methods. — Two  methods  of  performing  the  test 
will  be  described: 

(i)  Macroscopic    Method. — Separate    the    clot    and 

serum  in  the  capsule  by  centrifugation,  nick  the  wall  of 

.the  capsule  a  short  distance  above  the  serum  with  a  file, 

and  break  the  capsule  at  this  point.    Pipet  off  the  serum, 

placing  in  a  clean  test-tube,  and  add  9  volumes  of  salt 


5CX>  SERODIAGNOSTIC  METHODS 

solution.  Counting  the  drops  of  serum  as  they  fall  from 
the  capillary  pipet,  and  adding  nine  times  the  number  of 
drops  of  salt  solution  will  give  sufficiently  accurate 
dilution.  Now  place  a  number  of  very  small  test-tubes 
in  a  rack,  and  add  to  each  one  except  the  first  0.5  c.c.  of 
salt  solution  by  means  of  a  volumetric  pipet.  Then 
place  in  the  first  and  second  tubes  only  0.5  c.c.  of  the 
diluted  blood-serum.  Shake  the  second  tube,  and  with 
the  pipet  transfer  0.5  c.c.  to  the  third  tube.  Shake  this 
tube  and  transfer  0.5  c.c.  to  the  fourth  tube,  and  so  on, 
to  the  end.  Discard  0.5  c.c.  from  the  last  tube.  One 
tube,  to  serve  as  control,  should  contain  only  0.5  c.c.  of 
salt  solution,  without  any  serum.  The  volumetric 
pipet  should  be  thoroughly  rinsed  out  with  salt  solution 
after  each  transfer.  One  thus  arrives  at  a  series  of  dilu- 
tions of  the  serum,  as  follows:  i-io,  1-20,  1-40,  1-80, 
1-160,  1-320,  1-640,  etc.  Now  add  to  each  tube  a  like 
amount  (0.3  to  0.5  c.c.)  of  the  suspension  of  killed 
bacteria.  This  doubles  the  dilution  of  the  serum  in 
each  of  the  tubes.  Mix  all  the  tubes  thoroughly  by 
shaking,  and  place  the  rack  in  a  moderately  warm  place 
or  in  the  incubator  for  eight  to  twelve  hours.  In  those 
tubes  in  which  the  reaction  is  positive  there  will  be 
found  a  sediment  consisting  of  agglutinated  bacteria  at 
the  bottom  of  the  test-tube,  with  a  clear  supernatant 
fluid.  The  control  tube  and  the  negative  tubes  will  be 
cloudy  and  without  sediment. 

Dead  cultures  of  typhoid  bacilli,  together  with  all  appara- 
tus necessary  for  performing  the  macroscopic  test,  are  put 
up  at  moderate  cost  by  various  firms  under  the  names  of 
typhoid  diagnosticum,  typhoid  agglutometer,  etc.  Full 
directions  accompany  these  outfits. 


THE   WIDAL  REACTION  501 

Recently  Bass  and  Watkins  have  described  a  modification 
of  the  macroscopic  method  (using  very  concentrated  sus- 
pensions of  the  bacilli)  by  which  the  test  can  be  applied 
at  the  bedside.  Agglutination  occurs  within  a  few  minutes. 
The  apparatus  has  been  put  upon  the  market  by  Parke, 
Davis  &  Co. 

(2)  Microscopic  Method. — ^Arrange  a  series  of  dilu- 
tions of  the  blood-serum  as  above,  or,  if  dried  blood  is 
used,  macerate  the  dried  clot  with  salt  solution  or  tap- 
water.  In  the  latter  case,  unless  the  size  of  the  original 
drop  of  blood  is  known,  the  color  is  the  only  guide  as  to 
the  degree  of  dilution.  A  light  amber  color  will  roughly 
correspond  to  a  dilution  of  1-50.  From  such  a  dilution 
others  can  be  prepared.  On  the  center  of  each  of  several 
clean  cover-glasses  place  a  loopful  of  each  of  the  several 
dilutions,  employing  a  platinum  loop  of  about  2  mm. 
diameter.  With  the  same  loop  add  to  each  droplet  of 
diluted  serum  a  loop  from  a  twelve-  to  twenty-four-hour- 
old  bouillon  culture  of  the  organism  in  question,  or  of 
a  suspension  in  salt  solution  prepared  from  a  young 
agar-slant  culture.  This  doubles  the  dilution  of  serum 
in  each  case.  One  cover-glass  containing  no  serum 
should  be  prepared  to  serve  as  a  control.  Press  over 
each  cover-glass  a  hollow-ground  slide  previously  ringed 
with  vaselin.  Turn  the  slide  over  so  as  to  bring  the 
cover-glass  on  top.  Drying  is  prevented  and  the  cover- 
glass  held  in  place  by  the  vaselin. 

When  hollow-ground  slides  are  not  at  hand,  a  drop  each 
of  the  diluted  senmi  and  the  bacterial  suspension  may  be 
placed  in  the  center  of  a  heavy  ring  of  vaselin  on  an  ordinary 
slide  and  a  cover-glass  applied  to  this.  Vaselin  containing 
an  antiseptic  must  not,  of  course,  be  used  for  this  purpose. 


502  SERODIAGNOSTIC  METHODS 

Place  the  slides  in  a  moderately  warm  place  or  in  the 
incubator  at  37°  C.  for  two  hours.  Examine  under  the 
oil-immersion  lens  or,  better,  the  high-power  dry  lens 
of  the  microscope,  using  very  subdued  light.  Yellow 
(artificial)  light  gives  a  clearer  view  than  does  white 
Hght.  In  the  negative  slides  and  in  the  control  the 
organism  will  be  found  moving  freely  (if  a  motile  species) 
and  not  clumped;  while  in  the  positive  slides  the  organ- 
isms  are   found   motionless  and   gathered   in   tangled 


I 


;•  s  *. 


Fig.  175. — Showing  clumping  of  typhoid  bacilli  in  the  Widal  reaction.     At  one  point  a 
crenated  red  blood-corpuscle  is  seen  (Wright  and  Brown). 

masses  and  balls,  i.  e.,  they  are  agglutinated  (Fig.  175). 
Pseudoreactions,  in  which  there  are  a  few  small  clumps 
of  organisms  whose  motion  is  not  entirely  lost,  together 
with  many  freely  moving  organisms  scattered  through- 
out the  field,  should  not  mislead. 

3.  Interpretation  of  Results. — Experience  has  shown 
that  not  much  significance  attaches  to  reactions  occur- 
ring in  two  hours  with  dilutions  of  serum  less  than 
1-75  or  i-ioo.    With  killed  organisms  the  dilution  may 


BIOLOGIC   IDENTIFICATION    OF   UNKNOWN   PROTEINS    503 

be  somewhat  lower  than  when  living  organisms  are  em- 
ployed. On  the  other  hand,  recently  isolated  virulent 
cultures  are,  in  general,  more  resistant  to  agglutination 
than  old  attenuated  ones.  A  number  of  other  disease 
conditions  may  give  rise  to  a  positive  reaction  with  the 
typhoid  bacillus,  notably  infections  with  closely  related 
organisms,  such  as  the  colon  bacillus.  (In  such  cases, 
if  tests  are  made  with  several  species,  the  species  agglu- 
tinated in  the  highest  serum  dilution  may  generally,  but 
not  always,  be  regarded  as  the  cause  of  the  infection.) 
Agglutination  of  typhoid  bacilli  may  also,  though  rarely, 
occur  in  diseases  of  the  liver,  particularly  those  accom- 
panied by  jaundice,  and  in  pneumococcus  infections. 

In  typhoid  the  average  time  of  first  appearance  of 
the  reaction  in  the  dilutions  above  recommended  is  the 
fourteenth  to  the  fifteenth  day  of  actual  disease,  which 
corresponds  roughly  to  the  eighth  or  tenth  day  of  ap- 
parent disease.  In  doubtful  cases  the  test  should  be. 
repeated  at  frequent  intervals,  and  no  disappointment 
should  be  felt  if,  as  sometimes  (though  rarely)  happens, 
the  reaction  does  not  appear  until  the  twentieth  to  the 
twenty-fifth  day  of  the  disease.  It  is  evident,  there- 
fore, that  its  value  for  early  diagnosis  is  much  less 
than  that  of  the  blood-culture  (see  page  292).  After 
the  Widal  reaction  first  appears  it  remains  throughout 
the  whole  course  of  the  disease  and  often  persists  for 
years. 

B.  Biologic  Identification  of  Unknown  Proteins 

This  includes  the  differentiation  of  human  and  animal 
■  blood,  detection  of  meat  adulteration,  etc.,  by  means  of 
the  precipitin  test  (method  of  Uhlenhuth). 


504  SERODIAGNOSTIC  METHODS 

I.  Materials  Required. — The  following  equipment  is 
needed : 

(i)  Blood-serum  of  an  animal  (rabbit)  highly  im- 
munized against  the  protein  to  be  determined.  Im- 
munize several  rabbits  by  several  intravenous  or  intra- 
peritoneal injections  of,  for  example,  human  blood,  or 
better,  blood-serum.  Placental  blood  may  be  used,  or 
the  blood  may  be  obtained  as  for  the  Wassermann 
reaction.  The  doses  should  be  2  or  3  c.c.  and  should  be 
given  at  four-  or  five-day  intervals.  After  the  fourth  or 
fifth  dose  draw  2  or  3  c.c.  of  blood  from  an  ear  vein,  sep- 
arate the  serum,  and  determine  its  strength  as  follows: 

Prepare  dilutions  of  (in  this  case)  human  blood-serum 
in  the  proportions  i-iooo,  1-5000,  1-10,000,  1-20,000, 
etc.,  using  physiologic  salt  solution  as  a  diluent.  Place 
2  c.c.  each  of  the  several  dilutions  in  a  series  of  test-tubes. 
To  each  tube  add  o.i  c.c.  of  the  rabbit's  serum,  without 
shaking.  A  distinct  cloud  should  appear  in  the  lowest 
dilution  (i-iooo)  within  a  minute  or  two,  rapidly  deepen- 
ing to  a  heavy  flocculent  precipitate;  the  reaction  de- 
velops somewhat  more  slowly  in  the  higher  dilutions, 
but  no  reaction  is  significant  which  occurs  after  more 
than  twenty  minutes. 

If  the  titration  results  as  above  described,  anesthetize 
the  rabbit  while  it  is  in  a  fasting  condition,  as  otherwise 
the  serum  is  apt  to  be  opalescent;  remove  the  anterior 
breast  wall  under  aseptic  conditions;  take  out  the  left 
lung  and  open  the  heart,  so  as  to  allow  the  animal  to 
bleed  to  death  into  its  pleural  cavity.  Cover  the  body 
with  sterile  towels  wet  with  antiseptic  solution.  After 
clotting  has  occurred,  pipet  the  serum  into  sterile  bottles, 
and  add  y^  volume  of  5  per  cent.  carboUc  acid  as  a 


BIOLOGIC  IDENTIFICATION   OF  UNKNOWN  PROTEINS    505 

preservative.  If  the  serum  is  opalescent,  it  cannot  be 
used;  if  cloudy,  it  must  be  filtered  clear  through  a  sterile 
Berkefeld  filter.  Sometimes  the  cloudiness  can  be  re- 
moved by  simple  sedimentation.  The  titration  above 
described  should  be  repeated  and  verified  before  the 
serum  is  used  for  making  the  test  proper. 

Other  sera  immune  to  horse,  dog,  sheep,  beef,  fowl, 
etc.,  may,  of  course,  be  prepared  in  the  same  way. 

(2)  A  solution  of  the  unknown  substance  in  physio- 
logic salt  solution.  The  stock  dilution  should  be  about 
i-iooo.  If  made  from  a  dried  clot  this  can  only  be 
approximate.     The  following  criteria  may  be  used: 

(a)  It  should  be  almost  completely  colorless  by  trans- 
mitted light. 

(b)  It  should  give  only  a  slight  cloudiness  when 
heated  with  a  little  nitric  acid. 

(c)  It  should,  nevertheless,  foam  freely  on  shaking. 
The  solution  must  be  made  perfectly  clear — by  filtration 
if  necessary. 

2.  Method. — ^Arrange  a  series  of  test-tubes  and  charge 
them  as  follows: 

Tube  No.  I — 2  c.c.  of  the  unknown  solution  (diluted  i-iooo)  plus  o.i  c.c. 
of  immune  serum. 

Tube  No.  2 — 2  c.c.  of  normal  salt  solution  plus  o.i  c.c.  of  immune  seriun. 

Tube  No.  3 — 2  c.c.  of  a  i-iooo  dilution  of  known  serum  of  the  species 
corresponding  to  that  suspected  to  be  present  in  the 
unknown  material  plus  0.1  c.c.  of  immune  serum. 

Tube  No.  4 — 2  c.c.  of  a  i-iooo  dilution  of  a  serimi  from  a  species  differ- 
ent from  that  suspected  to  be  present  in  the  unknown 
material  plus  0.1  c.c.  of  immune  serum. 

Tube  No.  5 — 2  c.c.  of  the  unknown  solution  alone. 

"  When  the  first  and  third  tubes  give  a  positive  reac- 
tion, as  above  defined,  and  all  the  others  a  negative 


506  SERODIAGNOSTIC  METHODS 

reaction,  the  presence  of  the  protein  of  the  species 
tested  for  is  established.  It  must  be  remembered  that 
shaking  must  not  be  employed.  When  only  limited 
amounts  of  material  are  available,  the  test  can  be  made 
by  contact  in  capillary  tubes. 

Meat  adulteration  may  be  recognized  by  the  same 
method.  Usually  it  is  a  question  of  horse  flesh  sold 
as  beef  or  as  sausage.  Remove  about  30  gm.  of  the 
meat  from  the  deeper  portions  of  the  specimen  with  a 
clean  sterile  knife,  free  as  much  as  possible  from  fat, 
chop  up  on  a  clean  board,  and,  if  salted,  extract  several 
times  in  the  course  of  ten  minutes  with  sterile  distilled 
water.  Cover  the  30  gm.  of  freshened  chopped  meat 
with  about  50  c.c.  of  0.85  per  cent,  salt  solution,  and 
allow  it  to  stand  three  hours  at  room-temperature  or 
over  night  in  the  ice-box.  Pipet  off  the  supernatant 
fluid,  and  clarify  and  dilute  for  use  according  to  the 
criteria  given  above  for  preparing  extracts  of  the  un- 
known substance.  If  acid  to  Utmus,  it  is  to  be  neutral- 
ized before  use  by  adding  an  excess  of  an  insoluble 
alkali,  such  as  magnesium  oxid,  and  filtering. 

The  immune  serum  is  prepared  as  above  by  injecting 
rabbits  with,  in  this  case,  horse-scum.  It  must  have  a 
titer  of  at  least  1-20,000.  That  's,  V:  must  give  a  reac- 
tion with  the  homologous  serum  in  a  dilution  of  the 
latter  of  that  degree. 

3.  Interpretation  of  Results. — These  reactions  are 
very  closely  specific,  and  are  fully  established  for  foren- 
sic purposes.  Doubt  can  only  arise  as  between  the  pro- 
teins of  very  closely  related  species,  and  this  can  be 
practically  always  removed  by  the  use  of  adequate 
controls. 


OPSONINS  507 


C  Opsonins 


That  phagocytosis  plays  an  important  part  in  the 
body's  resistance  to  bacterial  invasion  has  long  been 
recognized.  According  to  Metchnikoff,  this  property  of 
leukocytes  resides  entirely  within  themselves,  depending 
upon  their  own  vital  activity.  The  studies  of  Wright 
and  Douglas,  upon  the  contrary,  indicate  that  the 
leukocytes  are  impotent  in  themselves,  and  can  ingest 
bacteria  only  in  the  presence  of  certain  substances  which 
exist  in  the  blood-plasma.  These  substances  have  been 
named  opsonins.  Their  nature  is  undetermined.  They 
probably  act  by  uniting  with  the  bacteria,  thus  preparing 
them  for  ingestion  by  the  leukocytes;  but  they  do  not 
cause  death  of  the  bacteria,  nor  produce  any  appreciable 
morphologic  change.  They  appear  to  be  more  or  less 
specific,  a  separate  opsonin  being  necessary  for  phago- 
cytosis of  each  species  of  bacteria.  There  are,  moreover, 
opsonins  for  other  formed  elements — red  blood-cor- 
puscles, for  example.  It  has  been  shown  that  the 
quantity  of  opsonins  in  the  blood  can  be  greatly  in- 
creased by  inoculation  with  dead  bacteria. 

To  measure  the  amount  of  any  particular  opsonin  in 
the  blood  Wright  has  devised  a  method  which  involves 
many  ingenious  and  delicate  technical  procedures. 
Much  skill,  such  as  is  attained  only  after  considerable 
training  in  laboratory  technic,  is  requisite,  and  there 
are  many  sources  of  error.  It  is,  therefore,  beyond  the 
province  of  this  work  to  recount  the  method  in  detail. 
In  a  general  way  it  consists  in:  (a)  Preparing  a  mixture 
of  equal  parts  of  the  patient's  blood-serum,  a  suspension 
of   the  specific  micro-organism,   and   a  suspension   of 


5o8  SERODIAGNOSTIC  METHODS 

washed  leukocytes;  (b)  preparing  a  similar  mixture, 
using  serum  of  a  normal  person;  (c)  incubating  both 
mixtures  for  a  definite  length  of  time;  and  (d)  making 
smears  from  each,  staining,  and  examining  with  an  oil- 
immersion  objective.  The  number  of  bacteria  which 
have  been  taken  up  by  a  definite  number  of  leukocytes 
is  counted,  and  the  average  number  of  bacteria  per 
leukocyte  is  calculated;  this  gives  the  "phagocytic  index." 
The  phagocytic  index  of  the  blood  under  investigation, 
divided  by  that  of  the  normal  blood,  gives  the  opsonic 
index  of  the  former,  the  opsonic  index  of  the  normal 
blood  being  taken  as  i.  Simon  regards  the  percentage 
of  leukocytes  which  have  ingested  bacteria  as  a  more 
accurate  measurement  of  the  amount  of  opsonins  than 
the  number  of  bacteria  ingested,  because  the  bacteria 
are  apt  to  adhere  and  be  taken  in  in  clumps. 

Because  of  its  simplicity  the  clinical  laboratory  worker 
will  prefer  some  modification  of  the  Leishman  method, 
which  uses  the  patient's  own  leukocytes.  It  is,  perhaps, 
as  accurate  as  the  original  method  of  Wright,  although 
variations  in  the  leukocyte  count  have  been  shown  to 
affect  the  result.  Two  pipets  like  those  shown  in  Fig. 
172  are  used. 


(i)  Make  a  suspension  of  the  specific  organism  by  mixing 
a  loopful  of  a  young  agar  culture  with  1  c.c.  of  a  solution  con- 
taining I  per  cent,  sodium  citrate  and  0.85  per  cent,  sodium 
chlorid.  Thoroughly  break  up  all  clumps  by  sucking  the 
fluid  in  and  forcing  it  out  of  one  of  the  capillary  pipets  held 
vertically  against  the  bottom  of  the  watch-glass. 

(2)  Puncture  the  patient's  ear,  wipe  off  the  first  drop  of 
blood,  and  from  the  second  draw  blood  into  the  other  pipet 


OPSONINS  509 

to  the  grease-pencil  mark,  let  in  a  bubble  of  air,  and  draw  in 
the  same  amount  of  bacterial  suspension. 

(3)  Mix  upon  a  slide  by  drawing  in  and  forcing  out  of  the 
pipet. 

(4)  Draw  the  mixture  high  up  in  the  pipet,  seal  the  tip 
in  the  flame,  and  place  in  the  incubator  for  fifteen  minutes. 

(5)  Repeat  steps  2,  3,  and  4  with  the  blood  of  a  normal 
person. 

(6)  After  incubation,  break  off  the  tip  of  the  pipet,  mix  the 
blood-bacteria  mixture,  and  spread  films  on  slides. 

(7)  Stain  with  Wright's  or  Harlow's  blood-stain  or  car- 
bol-thionin. 

(8)  With  an  oil-immersion  lens  count  the  bacteria  which 
have  been  taken  in  by  100  leukocytes,  and  calculate  the  aver- 
age number  per  leukocyte.  Divide  the  average  for  the  pa- 
tient by  the  average  for  the  normal  person.  This  gives  the 
opsonic  index.  If  in  the  patient's  blood  there  was  an  average 
of  4  bacteria  per  leukocyte,  and  in  the  normal  blood  5  bac- 
teria per  leukocyte,  the  opsonic  index  would  be  7  or  0.8. 

Wright  and  his  followers  regarded  the  opsonic  index 
as  an  index  of  the  power  of  the  body  to  combat  bacterial 
invasion.  They  claimed  very  great  practical  importance 
for  it  as  an  aid  to  diagnosis  and  as  a  guide  to  treatment 
by  the  vaccine  method.  This  method  of  treatment  con- 
sists in  increasing  the  amount  of  protective  substances 
in  the  blood  by  injections  of  normal  salt  suspensions  of 
dead  bacteria  of  the  same  species  as  that  which  has 
caused  and  is  maintaining  the  morbid  process,  these 
bacterial  suspensions  being  called  "vaccines."  Vaccine 
Therapy  (Chapter  IX)  has  taken  a  permanent  place 
among  our  methods  of  treatment  of  bacterial  infections, 
particularly  of  those  which  are  strictly  local,  but  the 


5IC  SERODIAGNOSTIC   METHODS 

opsonic  index  is  now  little  used  either  as  a  measure  of 
resisting  power  or  as  an  aid  to  diagnosis  and  guide  to 
treatment. 

IV.   REACTIONS    BASED    UPON    IMMUNE    BODIES    OF 
THE  THIRD  ORDER 

The  reactions  of  this  group  comprise  the  various 
applications  of  the  Wassermann  reaction  or,  more 
properly,  of  the  Bordet-Gengou  phenomenon  of  comple- 
ment-fixation or  deviation.  Since  the  reaction  involves 
three  active  substances — :viz.,  antigen  (the  substance 
inducing  the  immune  reaction) ;  the  specific  amboceptor, 
or  immune  substance;  and  the  non-specific  complement — 
it  is  possible  to  so  adjust  matters  that,  any  two  factors 
being  known,  the  third  may  be  determined  either  quali- 
tatively or  (roughly)  quantitatively.  Practically,  the 
method  is  employed  chiefly  for  determining  the  presence 
of  the  middle  term,  or  amboceptor.  It  may  be  applied  to 
the  diagnosis  of  any  disease  the  antigen  of  which  is 
known  and  which  can  be  obtained  in  suitable  form.  This 
includes  syphilis,  gonorrhea,  malignant  disease,  tuber- 
culosis, echinococcus,  cysticercus  infections,  trichiniasis, 
typhoid  and  pneumococcus,  meningococcus  and  sta- 
phylococcus infections,  etc.  In  several  of  these,  other 
and  simpler  methods  are,  however,  available.  The 
method  as  applied  to  the  first  four  diseases  above  men- 
tioned is  given  below. 

The  method  employed  is  based  upon  the  fact  that  if 
suitable  quantities  of  antigen,  amboceptor  (/.  e.,  patient's 
serum  containing  the  same),  and  complement  are  mixed 
together  and  warmed  gently  in  the  incubator,  a  sup- 
posedly chemical,  firm  union  of  the  three  takes  place. 


COMPLEMENT   DEVIATION    TEST  51I 

The  mere  fact  of  combining  in  this  way  produces, 
however,  no  visible  change  in  the  fiuid.  It  is  necessary, 
therefore,  to  test  for  free  complement  by  adding  the 
two  other  units  of  another  immune  system  which  also 
requires  the  presence  of  complement,  and  which  will 
produce  a  visible  reaction  if  free  complement  is  present. 
A  "hemolytic  system"  is  used  for  this  purpose.  The 
mixture  is  then  incubated  a  second  time.  If  the  three 
units  of  the  first  system  have  combined  (in  other  words, 
if  the  patient's  serum  contains  syphilis  antibody),  and 
not  otherwise,  the  complement  is  "fixed"  or  "deviated" 
during  the  first  incubation  period,  so  that  it  is  no  longer 
available  to  assist  in  completing  the  second  and  visible 
reaction  represented  by  the  hemolytic  system.  As  will 
be  seen  later,  an  elaborate  system  of  controls  is  needed. 

A.  Complement  Deviation  Test  for  Syphilis 

The  "Wassermann  Reaction 

Of  the  many  modifications  of  the  Wassermann  reac- 
tion, but  one,  the  standard  form  of  the  reaction,  will  be 
given. 

I.  Materials  Required. — The  following  reagents  are 
needed : 

(i)  Syphilitic  Antigen. — The  reaction,  originally  sup- 
posed to  depend  upon  the  presence  in  the  patient's 
serum  of  true  syphilis  antibodies,  is  now  known  to 
depend  instead  on  a  disorder  of  lipoid  metabolism  char- 
acterized by  the  presence  of  serum-foreign  lipoids 
in  the  serum.  Accordingly,  solutions  of  lipoids  from 
various  sources  can  be  used  for  the  test.  The  following 
may  be  recommended: 


512  SERODI AGNOSTIC   METHODS 

(a)  Grind  or  chop  the  liver  and  spleen  of  a  syphilitic 
fetus.  Place  in  a  suitable  vessel  and  add  4  to  10  parts 
of  absolute  ethyl  alcohol.  (The  amount  of  alcohol 
varies  in  the  hands  of  different  workers.)  Extract  for 
three  or  four  days  in  the  incubator  or  for  one  to  two 
weeks  at  room-temperature,  with  frequent  vigorous 
shakings.  Filter  through  paper.  The  filtrate  constitutes 
the  stock  solution,  which  is  diluted  with  salt  solution 
for  use,  as  described  later. 

(b)  Grind  in  a  mortar  with  quartz  sand  one  or  more 
guinea-pig  hearts,  previously  weighed,  place  in  a  suitable 
receiver,  and  add  10  c.c.  of  absolute  alcohol  for  each 
gram  of  heart  tissue.  Complete  the  preparation  as  above. 
This  solution  can  be  purchased  from  the  various  biologic 
houses. 

(2)  Antisheep  Amboceptor. — This  can  now  be  obtained 
so  readily  in  the  market  that  the  somewhat  elaborate 
method  of  preparation  may  be  omitted  here. 

(3)  Sheep's  Red  Blood-cells. — Where  a  slaughter-house 
is  available,  it  constitutes  the  most  convenient  source  of 
supply.  A  sterile  bottle  (about  loo-c.c.  capacity),  con- 
taining some  glass  beads,  bits  of  glass  rod,  or  steel 
shavings,  is  carried  to  the  slaughter-house.  After  the 
first  gush  of  blood  from  the  slaughtered  animal  has 
cleansed  the  wound,  the  bottle  is  filled  not  quite  full  with 
blood.  It  is  then  stoppered  and  the  bottle  kept  in  mo- 
tion for  ten  or  fifteen  minutes  or  until  defibrination  is 
complete.  For  use,  "wash"  the  cells  thoroughly  free 
from  serum  by  filling  centrifuge  tubes  about  one-quarter 
full  of  defibrinated  blood,  and  adding  0.9  per  cent  sodium 
chlorid  solution  to  the  top.  Centrifugate  thoroughly 
and  pipet  off  the  supernatant  fluid.      Again  fill  with 


COMPLEMENT  DEVIATION   TEST  513 

salt  solution,  mix,  centrifugate,  and  remove  the  super- 
natant fluid.  Repeat  at  least  three  times.  Finally, 
prepare  a  5  per  cent,  emulsion  by  adding  i  volume  of 
the  cells,  thoroughly  packed  by  centrifugation,  to  19 
volumes  of  salt  solution.  This  is  the  standard  against 
which  the  strength  of  all  other  solutions  is  measured 
or  titrated,  as  described  below. 

(4)  Complement.— Stun  a  fasting  guinea-pig  by  a  blow 
at  the  base  of  the  skull,  cut  the  throat,  and  collect  the 
blood  in  a  clean,  dry  dish.  The  serirai  may  be  allowed 
to  separate  spontaneously  over  night  in  the  ice-box,  or 
be  separated  just  before  use  by  centrifugation.  Serum 
more  than  twenty-four  hours  old  is  worthless  as  comple- 
ment. 

(5)  Patient's  Serum. — About  5  c.c.  of  blood  will  sufl&ce. 
A  convenient  method  consists  in  applying  an  Esmarch 
bandage  to  the  upper  arm,  after  cleansing  the  flexor 
surface  of  the  elbow  with  alcohol  or  tincture  of  iodin.  If 
the  patient  opens  and  closes  the  fist  vigorously  a  few 
times  the  veins  become  more  prominent.  Insert  the 
needle  of  the  syringe  above  described  above  or  alongside 
the  vein  and  at  an  acute  angle  to  the  skin  surface.  Once 
through  the  skin,  a  little  practice  will  enable  one  to 
quickly  find  the  way  into  the  vein.  Slow  withdrawal  of 
the  plunger  will  quickly  fill  the  syringe.  If  the  vein  is  a 
large  one  the  blood  will  flow  into  the  syringe,  driving  the 
plunger  ahead  of  it.  Remove  the  bandage  before  with- 
drawing the  syringe  to  avoid  a  hematoma.  Withdraw 
the  needle  quickly,  and  have  the  patient  or  an  assistant 
apply  fairly  firm  pressure  over  the  punctured  vein  for  a 
minute  or  two.  In  the  meantime  empty  the  syringe  into 
a  scrupulously  clean  test-tube,  and  immediately  wash. 

33 


514  SERODIAGNOSTIC  METHODS 

out  the  syringe  and  needle  thoroughly  with  water, 
followed,  especially  if  the  needle  is  of  steel,  by  alcohol. 
If  blood  is  given  time  to  clot  in  the  needle  or  syringe  the 
instrument  is  practically  ruined.  The  needle  should,  of 
course,  be  sterilized  by  boiling  before  use.  The  syringe 
should  be  clean  and  dry  (as  otherwise  hemolysis  will 
take  place),  but  need  not  be  sterilized. 

After  an  hour  or  two,  separate  the  clot,  if  necessary, 
from  the  test-tube  wall  with  a  clean  wire,  and  either 
complete  the  separation  of  the  serum  at  once  by  centrif- 
ugation  or  place  in  the  ice-box  over  night.  Transfer 
the  serum  with  a  capillary  pipet  to  a  second  clean  test- 
tube. 

Before  the  test  is  made  the  serum  is  "inactivated" 
ii.  e.,  the  native  complement  present  is  destroyed)  by 
immersing  the  tube  for  half  an  hour  in  the  water-bath 
at  55°  to  56°  C. 

Unless  a  considerable  number  of  sera  are  to  be  exam- 
ined simultaneously,  known  positive  and  known  negative 
control  sera  must  be  prepared  in  the  same  way. 

2.  The  Titrations. — The  strength  of  the  complement 
and  antisheep  amboceptor  must  be  determined  on  each 
occasion  of  its  use.  The  antigen  must  be  titrated  every 
few  weeks. 

(i)  Titration  of  the  Complement. — The  complement 
may  be  used  undiluted  or  in  varying  dilutions  of  from 
40  to  10  per  cent.  The  greater  the  dilution,  of  course, 
the  greater  the  accuracy  with  which  it  can  be  titrated. 
Assuming  that  it  is  to  be  used  in  a  40  per  cent,  dilu- 
tion (i  part  of  complement  serum  to  i|  parts  of  salt 
solution) ,  arrange  a  series  of  test-tubes  somewhat  as  fol- 
lows: 


COMPLEMENT   DEVIATION   TEST  515 

Tube  No.  I — 0.02  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  1 1  units  amboceptor.^ 
Tube  No.  2 — 0.04  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  I5  units  amboceptor. 
Tube  No.  3 — 0.06  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  I5  units  amboceptor. 
Tube  No.  4 — 0.08  c.c.  complement  serum  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  1 5  units  amboceptor. 
Tube  No.  5 — o.io  c.c.  complement  senim  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  15  units  amboceptor. 
Tube  No.  6 — 0.12  c.c.  complement  senun  plus  i  c.c.  5  per  cent,  sheep 

blood-cells  and  1 5  units  amboceptor. 

Make  up  all  tubes  to  a  like  volume  (1.5  or  2  c.c.)- 
Mix  thoroughly  by  gentle  shaking,  and  place  in  the 
incubator  (preferably  standing  in  a  dish  of  water,  since 
this  insures  rapid  and  uniform  heating  to  incubator 
temperature)  at  37°  C.  for  one  hour.  The  tube  contain- 
ing the  smallest  amount  of  complement  which  shows 
complete  solution  of  the  red  cells  (the  solution  bright 
red,  perfectly  clear,  and  free  from  sediment)  contains 
one  unit  of   complement.     A  slight  excess  over  this 

'  One  unit  of  amboceptor  is  the  amount  required  to  bring  about  solu- 
tion of  I  c.c.  of  the  5  per  cent,  red  cell  emulsion,  in  the  presence  of  i  unit 
of  complement,  in  one  hour  at  incubator  temperature.  In  the  same  way, 
I  unit  of  complement  is  the  amount  required  to  bring  about  solution  in 
the  presence  of  one  unit  of  amboceptor  imder  the  same  conditions.  In 
the  above  experiment  the  i|  imits  of  amboceptor  is  only  approximate. 
It  i?  assumed  that  the  worker  has  purchased  amboceptor  in  i-c.c.  vials, 
guaranteed  to  contain  1000  imits,  and  actually  containing  a  slight  excess 
over  that  amount.  For  use  this  is  diluted  with  loo  parts  of  salt  solution: 
0.1  c.c.  will  then  contain  something  over  i  unit.  On  the  first  occasion  of 
its  use,  0.15  c.c.  may  be  accepted  for  titration  purposes,  the  aim  being 
to  use  a  moderate  excess  to  allow  for  the  chance  of  deterioration  and 
slight  variations  in  the  strength  of  the  blood  emulsion.  On  each  later 
occasion  the  approximate  value  is  known  from  the  last  previous  titra- 
tion. Amboceptor  dilutions  keep  well  in  the  ice-box,  but  may  undergo 
a  very  abrupt  deterioration  at  the  end  of  about  six  months. 


5l6  SERODI AGNOSTIC  METHODS 

amount,  say  i|  units,  is  used  in  making  the  test  proper, 
to  allow  for  the  rapid  deterioration  which  takes  place 
and  for  the  small  amount  of  complement  directly  ab- 
sorbed by  the  antigen. 

(2)  Titration  of  the  Amboceptor. — Arrange  tubes  as 
follows : 

Red  cells  Amboceptor 

Tube.  Complement.  (s  per  cent.).  (i— 100  dilution). 

No.  I 1 2"  units '  i.o  c.c.  0.06  c.c. 

No.  2 1 1     "  1.0  c.c.  0.08  c.c. 

No.  3 1 5     "  1.0  c.c.  o.io  c.c. 

No.  4 15     "  1.0  c.c.  0.12  c.c. 

No.  5 1 5     "  I.o  c.c.  0.14  c.c. 

No.  6 15     "  1.0  c.c.  0.16  c.c. 

Bring  all  tubes  to  a  like  volume,  mix,  and  incubate  for 
one  hour.  The  tube  containing  the  smallest  amount 
of  amboceptor  which  causes  complete  hemolysis  contains 
one  unit.     Two  units  are  used  for  the  test  proper. 

(3)  Titration  of  the  Antigen. — The  stock  solution  is 
to  be  diluted  freshly  for  use  with  salt  solution.  This 
makes  a  milky  fluid.  The  amount  of  dilution  will 
vary  with  the  strength  of  the  stock  solution  as  deter- 
mined by  the  following  tests.  For  the  latter  a  lo  per 
cent,  dilution  may  be  employed. 

Arrange  test-tubes  as  follows: 

Antigen  Red  cells 

Tube.  (10  per  cent.).  (S  per  cent.). 

No.  I o. I  c.c.  I.o  ex. 

No.  2 0.2  c.c.  I.o  c.c. 

No.  3 0.3  c.c.  1.0  c.c. 

No.  4 0.4  c.c.  1.0  c.c. 

No.  5 0.5  c.c.  1.0  c.c. 

No.  6 0.6  c.c.  1.0  c.c. 

*  As  determined  in  the  previous  titration. 


COMPLEMENT  DEVIATION  TEST  517 

Bring  all  tubes  to  a  like  volume.  Mix  and  incubate. 
The  amount  used  in  making  the  test  proper  must  not  be 
more  than  one-half  the  smallest  amount  which  causes 
hemolysis  in  the  above.  A  modified  form  of  this  titra- 
tion is  repeated  each  time  the  antigen  is  used. 

Arrange  at  the  same  time  with  the  above  test-tubes 
as  follows: 

Antigen 

Tube.  (10  per  cent.).  Complement 

No.  I O.I  c.c.  i^  units 

No.  2 0.2  c.c.  ij     " 

No.  3 0.3  C.C.  i§     " 

No.  4 0.4  C.C.  li     " 

No.  5 0.5  C.c.  I J     " 

No.  6.. 0.6  c.c.  i|     " 

Bring  all  tubes  to  a  like  volume,  mix,  and  incubate. 
Then  add  to  all  tubes  i  c.c.  of  5  per  cent,  red  cell  emul- 
sion and  2  unit^  amboceptor  solution.  Mix  and  reincu- 
bate.  If  the  antigen  is  "anticomplementary"  it  will 
prevent  hemolysis  in  one  or  more  of  the  tubes.  The 
amount  used  for  the  test  proper  must  not  exceed  one- 
half  the  smallest  amount  showing  such  action. 

The  antigen  must  also  be  shown  to  react  with  known 
positive  sera,  and  the  amount  required  to  produce  a 
reaction  determined.  For  this  purpose  an  abundant 
supply  of  serum  from  a  patient  with  active  secondary 
S)q3hilis  (still  better,  from  several  such  patients)  is  ob- 
tained, and  the  complete  reaction  carried  out  as  described 
below,  employing  varying  amounts  of  the  antigen  dilu- 
tion, e.  g.,  0.04,  0.06,  0.08,  0.1,  0.12,  0.14  c.c,  etc.  For 
the  test  proper  an  amount  is  used  in  the  greatest  pos- 
sible excess  of  that  amount  which  gives  a  positive  re- 
action, but  which  complies,  however,  with  the  require- 


5l8  SERODIAGNOSTIC  METHODS 

ments  mentioned  above  as  to  hemolytic  and  anticom- 
plementary action. 

3.  Errors  and  Their  Causes. — (i)  Dirty  glassware 
unquestionably  is  responsible  for  most  of  the  errors. 
No  control  can  eliminate  an  error  caused  by  a  single 
dirty  test-tube  among  a  hundred  clean  ones.  The  hemo- 
lytic system  will  be  completely  destroyed  in  such  a  tube, 
with  the  Ukelihood  of  interpreting  the  result  as  a  positive 
reaction,  or  as  due  to  an  anticomplementary  patient's 
serum,  etc.,  depending  on  what  it  happens  to  be  used  for. 
Glassware  need  not  be  sterile,  but  must  be  absolutely 
clean.  Never  allow  used  tubes  to  stand  and  dry  out. 
Immediately  the  work  is  finished  wash  with  soap  and 
water,  rinse  thoroughly  with  clean  water  followed  by 
dilute  (10  per  cent.)  nitric  acid,  then  caustic  soda  solu- 
tion, then  several  changes  of  distilled  water.  Place  in  a 
basket  and  dry  in  a  dry-air  sterilizer  if  one  is  available. 
New  glassware  should  be  prepared  for  use  in  the  same 
way.  Tubes  may  be  kept  stored  in  the  oven  or  set 
aside  in  a  clean  dust-proof  cupboard.  Tubes  used  for 
this  purpose  should  never  be  used  for  any  other.  Other 
glassware  should  be  cleaned  with  equal  care,  and  pre- 
served from  any  other  use. 

(2)  The  patient's  serum  may  be  "anticomplementary," 
i.  e.,  it  may  have  the  power  to  combine  with  or  absorb 
complement  in  the  absence  of  antigen.  Serum  which 
has  been  kept  too  long,  or  which  has  been  inactivated 
at  a  temperature  above  56°  C,  is  apt  to  exhibit  this 
property.  The  anticomplementary  property  may  some- 
times be  made  to  disappear  by  renewed  inactivation. 
If  this  fails,  the  serum  must  be  discarded.  A  control  of 
this  property  is  included  in  setting  up  the  test. 


COMPLEMENT  DEVIATION   TEST  519 

(3)  The  antigen  may  become  anticomplementary  or 
hemolytic,  or  both.  When  this  happens  it  must  be  dis- 
carded. A  control  for  this  is  set  up  each  time  the  antigen 
is  used. 

(4)  The  hemolytic  system  may  fail  to  function  for  a 
variety  of  reasons.  A  control  of  this  is  furnished  by 
the  titration  of  complement  and  amboceptor  above 
described. 

4.  The  Test  Proper. — Assuming  for  the  moment  that 
all  preparations  have  been  completed  and  the  titra- 
tions satisfactorily  performed,  one  proceeds  to  set  up 
the  test  proper.  We  may  suppose  that  we  are  dealing 
with  at  least  three  sera — viz.,  the  patient's  serum  and 
known  positive  and  known  negative  controls.  Arrange 
in  a  rack  having  two  rows  of  holes  test-tubes  as  shown 
on  p.  520. 

Bring  all  tubes  to  a  hke  volume  (1.5  or  2  c.c.)  by  the 
addition  of  salt  solution.  Mix  by  gentle  shaking  and 
place  in  the  incubator  for  one  hour.  The  rack  should 
stand  with  the  tubes  immersed  in  water  to  about  the 
level  of  the  contents.  Then  add  to  all  the  tubes  except 
the  last  one,  already  containing  blood-cells,  2  units  of 
antisheep  amboceptor  dilution  and  i  c.c.  of  5  per  cent. 
sheep  red  cell  emulsion.  Mix  as  before  and  incubate  for 
two  hours.  The  tubes  in  the  back  row  show  for  each 
serum  tested  whether  any  of  them  is  anticomplementary. 
They  should  all  show  complete  solution  of  the  red  cells. 
The  last  two  tubes  show  whether  the  antigen  is  hemolytic 
or  anticomplementary  respectively.  The  first,  contain- 
ing complement,  should  show  complete  hemolysis.  The 
"second,  containing  only  antigen  and  red  cells,  should 
show  no  solution.    Assuming  that  these  controls  are  all 


520 


SERODIAGNOSTIC  METHODS 


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COMPLEMENT   DEVIATION   TEST  521 

satisfactory,  one  turns  to  the  tubes  in  the  front  row. 
The  known  positive  control  shows  no  solution  of  the  red 
cells,  the  complement  having  been  deviated  or  bound 
during  the  first  incubation,  and  hence  being  not  available 
for  the  reaction  with  the  red  cells.  For  the  opposite 
reason  the  known  negative  senun  will  show  complete 
solution.  The  unknown  serum  will  behave  like  the  first 
or  the  second,  according  as  it  is  positive  or  negative. 

It  is  apparent  that  an  excess  of  complement  may  con- 
vert a  positive  reaction  into  a  negative,  while  a  defi- 
ciency may  cause  a  negative  serum  to  behave  like  a 
(more  or  less)  positive  one.  It  is  to  avoid  this  contin- 
gency that  the  unknown  quantity  of  complement  present 
in  the  fresh  patient's  serum  is  removed  by  inactivation, 
to  be  replaced  by  an  accurately  measured  amoimt  of 
guinea-pig  complement. 

With  cerebrospinal  fluid  the  reaction  is  carried  out 
in  the  same  way,  except  that  this  fluid  must  not  he  inac- 
tivated, and  is  used  in  larger  amounts.  When  enough 
fluid  is  available  the  test  should  be  set  up  so  as  to  give 
a  reading  for  0.4,  0.6,  0.8,  and  i  c.c.  When  economy 
of  material  is  necessary  a  reading  should  be  obtained 
for  0.5  and  i  c.c.  Further  modifications  may,  of 
course,  be  imposed  by  the  exigencies  of  the  case. 

Several  degrees  of  the  reaction  are  recognized  and  are 
customarily  indicated  as  follows: 

Complete  inhibition  (cells  intact  with  colorless 

supernatant  fluid),  -I-+-1-+  or  4+. 

Almost  complete  inhibition,  +++  or  3  -|-. 

About  one-half  complete  inhibition,  -H+  or  2  -H. 

Slight  inhibition,  -f  or  i  -h. 

No  inhibition.  o- 


522  SERODI AGNOSTIC  METHODS 

5.  Interpretation  of  Results. — (i)  Jaundice  and  marked 
alcoholism  may  convert  a  positive  reaction  into  a  nega- 
tive one. 

(2)  Scarlet  fever,  leprosy,  active  malaria,  and  malig- 
nant tumors  may  cause  a  positive  reaction, 

(3)  The  reaction  is  negative  in  primary  syphilis,  but 
becomes  rapidly  and  strongly  positive  as  the  general 
manifestations  of  the  disease  develop.  During  this  stage 
only  a  strongly  positive  reaction  should  be  regarded  as 
significant.  In  late  and  especially  in  latent  syphilis 
the  reaction  again  grows  weaker.  More  significance 
may,  therefore,  attach  to  weak  reactions  in  such  cases. 

(4)  A  positive  reaction  quickly  becomes  negative 
under  specific  treatment,  to  recur  if  treatment  is  ineffi- 
cient. Apparently  cured  cases  may  show  a  positive 
reaction  six  months  to  a  year  after  a  "provocative"  dose 
of  salvarsan. 

(5)  The  behavior  of  the  blood  is  no  guide  as  to  the 
condition  of  the  central  nervous  system.  Recent  investi- 
gations have  shown  that  the  central  nervous  system 
becomes  involved  very  early  in  practically  all  cases, 
and  the  organisms  so  located  are  pecuUarly  inaccessible 
to  attack  by  present  methods.  No  case  may  be  re- 
garded as  cured  until  both  blood  and  cerebrospinal 
fluid  show  a  persistent  normal  condition. 

Routine  Methods. — The  labor  involved  in  carrying  out  the 
somewhat  elaborate  details  of  the  method  as  above  outlined 
may  be  materially  lightened  by  systematizing  the  work 
somewhat  as  follows:  On  the  day  before  the  tests  are  to 
be  made  prepare  an  abundance  of  clean  glassware  and  the 
red  cell  emulsion;  see  that  the  water-bath  is  properly  reg- 


COMPLEMENT   DEVIATION   TEST  523 

ulated;  and,  if  desired,  bleed  one  or  more  guinea-pigs  for 
complement,  and  place  the  blood  in  the  ice-box.  In  the 
morning  proceed  as  follows: 

(i)  Set  up  the  complement  titration,  place  in  the  incuba- 
tor, and  mark  the  time. 

(2)  Pipet  off  the  sera  to  be  tested  into  clean  test-tubes, 
and  place  in  the  water-bath  to  inactivate.    Mark  the  time. 

(3)  Arrange  in  the  rack  the  tubes  needed  for  the  test,  the 
antigen  control,  and  the  amboceptor  titration. 

(4)  By  the  time  inactivation  is  complete  the  complement 
titration  will  be  nearly  or  quite  finished.  Forty-five  minutes 
will  suffice  for  the  latter.  Now  set  up  the  tests  proper,  with 
the  controls  and  the  amboceptor  titration. 

(5)  At  the  end  of  the  hour  the  titration  may  be  read,  and 
the  indicated  amount,  and  the  red  cells,  added  to  all  tubes. 
Two  hours  later  the  final  result  is  read  and  recorded.  Glass- 
ware should  immediately  be  washed  and  put  away  for  the 
next  occasion.  A  little  experience  will  enable  one  to  make 
from  twenty-five  to  fifty  tests  between  9  A.  m.  and  3  P.  m. 

B.  Complement  Deviation  Test  for  Gonorrhea 

Method  of  Schwartz  and  McNeil 

The  method  as  given  below  represents  minor  modifi- 
cations of  the  original  method  suggested  by  experience 
in  the  writer's  laboratory.  The  antigen  is  an  autolysate 
of  a  large  number  of  strains  of  the  gonococci.  It  may  be 
obtained  from  Parke,  Davis  &  Co.  For  use,  dilute  with 
9  parts  of  salt  solution.  The  amount  used  for  the  test 
is  one  which  gives  a  strong  positive  reaction  with  a  known 
positive  serum  or  with  the  antigonococcic  senmi  of  Tor- 
jey  (also  marketed  by  Parke,  Davis  &  Co.),  provided  this 
amount  is  not  anticomplementary.  In  our  experience  0.15 
c.c.  of  a  10  per  cent,  dilution  has  met  these  conditions. 


524  SERODIAGNOSTIC  METHODS 

The  complement  is  used  in  a  lo  per  cent,  dilution. 
Complement  and  amboceptor  are  titrated  against  o.i 
c.c.  of  5  per  cent,  sheep  cell  emulsion,  instead  of  i  c.c. 
The  same  quantity  of  red  cell  emulsion  is,  of  course, 
also  used  in  making  the  test. 

The  patient's  serum  is  used  inactivated.  In  the  orig- 
inal method  the  test  is  carried  out  with  0.05,  o.io,  and 
0.15  c.c.  In  our  experience  0.05  c.c.  is  almost  invariably 
negative,  while  0.15  c.c.  is  almost  invariably  anticom- 
plementary.   We  have,  therefore,  used  only  o.i  c.c.^ 

In  other  respects  the  test  is  carried  out  exactly  like 
the  test  for  syphiHs.  The  reaction  is  negative  during 
the  acute  stages  of  the  disease,  but  is  useful  in  deter- 
mining the  presence  of  a  focus  of  chronic  infection.  Its 
chief  importance  lies  in  the  fact  that  it  becomes  negative 
in  a  short  time  (probably  about  two  weeks)  after  a  cure 
is  completed. 

C  Complement  Deviation  Test   for  Malignant 
Disease 

Method  of  von  Dungem 

I .  The  antigen  is  prepared  in  either  of  the  two  follow- 
ing ways : 

(a)  Free  tissue  from  a  malignant  tumor  as  much  as 
possible  of  fat  and  necrotic  tissue.  Chop  up  and  ex- 
tract for  one  or  two  weeks  with  20  volumes  of  chemically 
pure  acetone,  with  frequent  shaking.  Filter.  For  use, 
evaporate  a  portion  to  dryness  at  37°  C.  and  redissolve 
in  half  the  amount  of  absolute  alcohol.     Dilute  with 

1  My  assistant,  Dr.  T.  F.  Walker,  has  kindly  furnished  me  with  these 
data,  based  on  an  unusually  extensive  experience  in  my  laboratory  with 
the  method. 


COMPLEMENT  DEVIATION   TEST  525 

salt  solution  according  to  the  result  of  the  titration. 
The  dose  for  the  test  is  the  largest  possible,  provided 
that  twice  the  amount  is  not  anticomplementary.  The 
dilution  is  arranged  simply  to  contain  the  required 
amount  in  a  convenient  volume  unit,  e.  g.,  0.5  c.c. 

(b)  Receive  10  c.c.  of  himian  blood  (preferably  from 
a  general  paralytic)  in  a  graduated  container  containing 
0.1  c.c.  of  a  2  per  cent,  sodium  oxalate  solution.  Wash 
the  cells  at  least  three  times  with  normal  salt  solution. 
After  the  last  centrifugation,  which  should  be  as  com- 
plete as  possible,  weigh  the  cells.  This  can  be  done 
readily  by  difference  if  the  weight  of  the  centrifuge  tube 
or  tubes  is  known.  Add  20  volumes  of  chemically  pure 
acetone  for  i  part  by  weight  of  the  cells.  Extract  for 
three  days  at  room  temperature,  with  occasional  shak- 
ing. Filter,  and  evaporate  the  filtrate  in  a  weighed 
dish  to  dryness  in  the  incubator.  Take  up  the  residue 
with  enough  96  per  cent,  alcohol  to  make  a  i  per  cent, 
solution.  The  residue  will  dissolve  completely  if  the 
alcohol  is  added  as  soon  as  the  acetone  evaporates. 
For  use,  mix  i  part  of  the  alcoholic  solution  slowly  with 
4  parts  of  salt  solution,  and  shake  thoroughly.  Titrate 
as  above.  About  0.8  c.c.  is  the  usual  dose  for  the  reac- 
tion. This  form  of  antigen  is  preferred  by  von  Dimgem 
to  the  cancer  extract  described  above. 

2.  The  serum  to  be  tested  (and  when  possible,  positive 
and  negative  controls)  is  obtained  aseptically,  and  after 
separation  from  the  clot  should  be  kept  two  or  three 
days  in  the  ice-box  before  completing  the  test.     Then 

add  2  volumes  of  —  caustic  soda  solution,  chemically 

pure  and  kept  free  from  absorbing  carbonic  acid  gas 


526  SERODIAGNOSTIC  METHODS 

from  the  air,  and  prepared  as  needed  by  diluting  one 

N 
volume  of  —  soda  with  four  volumes  of  normal  salt 

solution,  and  inactivate  for  one-half  hour  at  54°  C. 

3.  The  complement  serum  is  used  without  titration, 
I  c.c.  of  a  5  per  cent,  solution. 

4.  Von  Dungern  uses  a  beef  hemolytic  system.  I  have 
found  the  customary  sheep  system  quite  as  applicable. 

Arrange  two  rows  of  test-tubes,  in  each  pair  of  which, 
front  and  rear,  place  0.6,  0.3,  0.15,  0.075  c-c,  respectively, 
of  the  inactivated  soda-serum  mixture.  To  all  tubes 
add  I  c.c.  of  5  per  cent,  complement  and  to  the  front  row 
add  the  titrated  amount  of  antigen — e.g.,  0.8  c.c.  Bring 
all  the  tubes  to  a  like  volume,  mix,  and  expose  for 
three  hours  at  room  temperature.  Add  to  all  tubes  2 
units  of  amboceptor  (i  unit  being  the  smallest  amount 
which  completely  hemolyses  i  c.c.  of  5  per  cent,  red  cell 
emulsion  in  two  hours  in  the  incubator  in  the  presence 
of  I  c.c.  of  5  per  cent,  complement  dilution)  and  i  c.c. 
of  5  per  cent,  red  cell  emulsion.  The  results  are  read 
after  standing  for  three  hours  at  room- temperature. 

Inactivation  eliminates  heterologous  reactions  due  to 
syphilis,  and  the  addition  of  the  alkali  eliminates  similar 
errors  due  to  tuberculosis.  A  correct  result  may  be 
expected  in  over  90  per  cent,  of  the  cases. 

D.  Complement  Deviation  Test  for  Tuberculosis 

Method  of  Hammer 

The  antigen  is  a  mixture  of  Koch's  old  tuberculin 
and  an  extract  of  tuberculous  granulation  tissue  freed 
as  much  as  possible  from  other  tissue.    Tissues  from  a 


COMPLEMENT   DEVIATION    TEST  527 

surgical  lesion,  such  as  the  knee,  are  most  suitable. 
Cover  the  tissue  with  4  parts  of  alcohol  and  extract  for 
three  to  five  days.  Filter,  and  dilute  the  filtrate  with 
3  parts  of  salt  solution  for  use.  Test  0.4,  0.2,  and 
0.1  c.c.  of  this  against  o.i  c.c.  of  known  positive  serum. 
Or,  cover  the  tissue  with  9  parts  of  acetone  and  extract 
for  ten  days.  Filter,  and  evaporate  to  dryness  at  37°  C. 
Take  up  the  residue  in  an  equal  volume  of  alcohol  and 
dilute  for  use  with  10  volumes  of  salt  solution.  Titrate 
as  above.  In  either  case  the  dose  used  is  the  largest, 
twice  which  is  not  anticomplementary. 

Now  add  to  9  volumes  of  the  diluted  extract  i  volume 
of  old  tuberculin,  and  repeat  the  titration  as  above. 
The  dose  is  determined  according  to  the  same  rule. 
A  certain  proportion  of  cases  will  react  with  one  or 
other  of  the  antigens  alone,  but  the  larger  percentage 
of  positive  results  will  be  obtained  with  the  mixed 
antigen. 

Arrange  the  tubes  as  for  the  Wassermann  reaction. 
In  all  the  tubes  place  i  c.c.  of  5  per  cent,  complement 
serum.  To  the  front  tubes  add  the  titrated  dose  of  anti- 
gen. In  each  pair  of  tubes,  front  and  rear,  place  o.i  c.c. 
of  the  several  sera  respectively,  inactivated  at  56°  C. 
for  thirty  minutes.  Bring  all  the  tubes  to  a  Hke  volume, 
mix,  and  let  stand  for  three  hours  at  room- temperature. 
Add  2  units  of  amboceptor  and  i  c.c.  of  5  per  cent,  red 
cell  emulsion.  Mix,  and  place  in  the  incubator  for  one 
hour.     The  tests  are  then  ready  for  the  final  reading. 

-  The  three  methods  which  follow  do  not  rest  on  any  of 
the  laws  of  immunity  above  mentioned.  They,  there- 
fore, must  be  classified  separately. 


528  SERODIAGNOSTIC   METHODS 

V.  COBRA- VENOM  TEST  FOR  SYPHILIS 
Method  of  Weil 

Of  the  several  cobra-venom  reactions,  the  method  of 
Weil,  for  the  diagnosis  of  syphilis,  possesses  the  greatest 
practical  value,  and  is  here  given.  It  appears  to  depend 
upon  the  same  disturbance  of  Upoid  metabolism  which  is 
responsible  for  the  Wassermann  reaction.  It  is  known 
that  syphilis  is  characterized  by  a  withdrawal  of  lipoids 
from  their  chief  depots,  viz.,  the  central  nervous  system 
and  the  red  blood-cells,  with  a  marked  increase  of  the 
same  in  the  fluid  part  of  the  blood.  Since  it  is  also 
known  that  the  hemolytic  action  of  the  cobra  venom 
depends  upon  its  activation  by  lecithin,  in  other  words, 
upon  a  lecithin-venom  complex  in  which  the  lecithin 
serves  as  complement,  it  may  fairly  be  assumed  that 
the  loss  of  lipoids  by  the  red  cells  is  responsible  for  the 
increased  resistance  to  hemolysis  by  cobra  venom  upon 
which  Weil's  reaction  is  based. 

I.  Materials  Required. — (i)  The  cobra  venom  may  be 
obtained  from  Poulenc  Freres,  Paris.  Weil's  stock 
solution  is  a  0.5  per  cent,  solution  in  0.9  per  cent,  salt 
solution,  made,  of  course,  very  accurately.  It  deterior- 
ates very  rapidly  unless  kept  frozen.  For  this  reason 
I  have  tried  very  successfully  the  solvent  usually  em- 
ployed for  the  purpose  of  other  reactions  in  this  group, 
viz.,  a  I  per  cent,  solution  of  venom  in  equal  parts  of 
distilled  water  and  chemically  pure  glycerin.  Before  it 
is  used,  this  should  be  allowed  to  stand  several  days  in 
the  ice-box,  where  it  keeps  extraordinarily  well. 

(2)  The  blood-cells  to  be  tested.  'Have  ready  normal 
salt  solution  to  which   2  per  cent,   sodium  citrate  is 


COBRA- VENOM   TEST   FOR   SYPHILIS  529 

freshly  added,  and  which  has  been  cooled  in  the  ice- 
box. Into  about  lo  c.c.  of  this,  contained  in  a  graduated 
centrifuge  tube,  discharge  about  2  c.c.  of  the  patient's 
blood.  Do  not  shake.  Wash  at  least  four  times  with 
0.9  per  cent,  salt  solution.  The  last  washing  of  all 
bloods  in  a  series  is  done  at  the  same  speed  and  for  the 
same  length  of  time.  Accurate  and  uniform  dilution  of 
the  cells  is,  of  course,  an  absolute  essential  to  obtain 
comparable  readings.  Pipet  off  the  last  wash-water  and 
make  up  to  a  4  per  cent,  emulsion  by  adding  24  vol- 
umes of  solution  to  the  i  volmne  of  ceUs  as  read  in  the 
graded  tube  before  they  are  disturbed.  The  salt  solu- 
tion used  for  washing  and  diluting  should  be  ice  cold 
and  the  final  emulsion  should  be  placed  on  ice  several 
hours  before  the  test  is  made. 

2.  Method. — From  the  stock  solution  of  venom  pre- 
pare the  following  solutions  for  the  test:  1-10,000, 
1-20,000,  1-30,000,  1-40,000.  Arrange  a  suitable  rack 
with  4  tubes  for  each  test.  In  the  respective  tubes  of 
each  row  place  i  c.c.  of  the  several  venom  solutions  and 
I  c.c.  of  the  cell  emulsion.  Incubate  for  one  hour  at 
37°  C.  Mix  thoroughly  by  gentle  shaking  and  place  in 
the  ice-box  over  night.  In  the  morning  again  mix  thor- 
oughly and  make  the  final  reading  an  hour  later.  The 
result  wiU  depend  on  comparison  with  known  normal 
cells.    Something  like  the  following  may  be  anticipated: 

No  hemolysis  at  1-10,000  =  strongly  positive. 

Moderate  hemolysis  at  1-20,000  =  positive. 

Partial  hemolysis  at  1-30,000  =  negative. 

Complete  hemolysis  at  1-40,000  =  hypersensitive. 

The  test  appears  later  in  the  disease  than  the  Wasser- 
mann  reaction,  and  yields  a  higher  percentage  of  positive 
34 


53©  SERODIAGNOSTIC  METHODS 

results  in  late  latent  syphilis.  Furthermore,  it  yields 
less  quickly  to  treatment.  It  is  unquestionably  an  im- 
portant aid  to  diagnosis  and  treatment  in  the  class  of 
cases  indicated. 

VL  THE  PROTECTIVE  FERMENT  REACTIONS 
Method  of  Abderhalden 

The  reaction  depends  upon  the  following  laws,  briefly 
stated : 

When  a  foreign  protein,  fat,  or  carbohydrate  is  in- 
jected directly  into  the  circulation,  ferments,  which  are 
able  to  digest  the  material  injected,  promptly  appear  in 
the  circulation.  These  ferments  are  specific,  i.  e.,  they 
act  only  upon  the  substance  the  presence  of  which 
has  led  to  their  appearance  in  the  blood. 

The  cells  of  the  body  are  vital  units,  and  carry  out 
with  a  certain  measure  of  independence  each  a  series 
of  metabolic  changes  peculiar  to  itself.  The  body  fluids, 
and  perhaps  the  body  cells,  are  adapted  to  dispose  of 
metabolic  products  only  when  they  receive  them  in  a 
certain  state,  which  may  be  regarded  as  the  normal. 
If  the  cells  of  any  organ  discharge  into  the  blood  any 
product  in  a  form  to  which  the  blood  is  not  accustomed, 
such  product  behaves  as  a  foreign  substance.  There 
results  the  concomitant  appearance  of  the  ferment 
above  mentioned,  and  this  whether  the  substance  is  a 
normal  product  of  the  cell  which  normally  does  not  enter 
the  blood,  or  whether  it  is  the  product  of  diseased  and 
abnormal  metaboUsm,  and,  therefore,  equally  foreign  to 
both  cell  and  blood.  A  substance  may,  therefore,  be 
foreign  to  the  organism  as  a  whole,  yet  normal  to  many 
of  its  parts — normal  to  the  liver,  for  example,  and  for- 


THE   PROTECTIVE   FERMENT   REACTIONS  53 1 

eign  to  every  other  tissue.  Hence,  a  distinction  must  be 
made  between  body-native  and  blood-native,  organ- 
foreign  and  blood-foreign,  etc. 

The  source  of  the  ferments  is  unknown.  They  appear 
too  quickly  to  permit  the  assumption  that  they  originate 
by  a  process  analogous  to  that  which  produces  the  im- 
mune substances.  Abderhalden  has  recently  advanced 
the  hypothesis  that  they  are  secreted  by  the  same  cells 
which  produce  the  substance  to  be  digested,  or,  in  the 
case  of  parenteral  administration  of  body-foreign  sub- 
stances, by  the  cells  producing  the  ferment  which, 
under  normal  conditions,  would  digest  the  substance 
in  the  intestinal  tract.  I  would  add  that  in  the  former 
case  the  ferments  may  then  be  identical,  in  part  at  least, 
with  those  responsible  for  autolysis. 

It  may  be  flatly  asserted  at  the  outset  that,  in  spite 
of  the  severe  criticism  to  which  the  method  has  been 
subjected,  the  correctness  of  Abderhalden's  thesis  has 
been  abundantly  confirmed.  In  every  case  results  which 
seem  to  show  the  contrary  have  been  shown  to  depend  on 
faulty  technic.  The  technical  difficulties  involved  are 
very  great.  In  the  following  paragraphs  I  shall,  for 
the  sake  of  brevity,  content  myself  with  pointing  out 
the  precautions  which  must  be  rigidly  observed,  without 
pausing  for  the  why's,  merely  reminding  the  reader  of 
Abderhalden's  dictum,  that  no  one  is  competent  to  ex- 
press an  opinion  as  to  the  method  who  has  not  first  con- 
vinced himself  of  his  ability  to  obtain  invariably  correct 
results,  to  distinguish  invariably,  for  example,  between 
pregnant  and  non-pregnant  individuals. 

A  special  room  must  be  provided  with  special  appara- 
tus, including  glassware,  sterilizer,  incubator,  etc.,  not 


532  SERODIAGNOSTIC   METHODS 

to  be  used  for  any  other  purpose.  All  utensils  must  be 
rigidly  clean,  as  for  the  Wassermann  reaction,  and,  in 
addition,  must  be  sterilized  as  for  bacteriologic  work. 
Two  methods  are  available,  which  may  be  used  alone 
or  in  combination: 

A.  The  Dialyzation  Method 

This  method  depends  upon  the  fact  that  albumin  is 
not  dialyzable,  while  peptone  and  simpler  products 
produced  from  it  by  digestion  are  dialyzable. 

I.  Preparation  of  Materials. — (i)  The  Substrate. — 
The  preparation  of  all  organs  is  the  same,  except  that 
material  rich  in  fat  and  lipoids,  such  as  the  liver,  kidney, 
brain,  and  tubercle  bacillus,  must,  in  addition,  be  ex- 
tracted with  carbon  tetrachlorid  in  a  Sohxlet  apparatus. 
Placental  tissue  is  obtained  fresh,  other  organs  are 
obtained  from  autopsy  material,  preferably  from  cases 
of  death  by  violence.  The  pathologic  condition  of  organs 
used  should  be  determined  by  careful  examination, 
microscopic  and  otherwise. 

Preparation  of  placental  tissue  may  be  taken  as  the 
type.  Remove  visible  blood-clots,  together  with  the 
membranes,  cut  into  small  pieces,  and  squeeze  them 
out  hard  in  running  water  (if  the  tap-water  is  soft  it 
is  better  to  use  normal  salt  solution).  The  easiest  way 
is  to  place  the  tissue  on  a  sieve,  and  allow  the  water 
to  run  over  it  uninterruptedly,  squeezing  the  individual 
pieces.  In  the  case  of  bacteria  and  finely  divided  tissue, 
a  centrifuge  must  be  substituted  for  the  sieve.  From 
time  to  time  place  in  a  cloth  and  wring  out.  Discard 
pieces  containing  clotted  blood  and  as  much  of  the  con- 
nective tissue  as  possible.    Finally,  crush  the  tissue  in 


THE  PROTECTIVE   FERMENT   REACTIONS  533 

a  mortar  and  wash  again  in  running  water.  The  tissue 
finally  becomes  perfectly  white,  except  liver,  spleen,  and 
kidney,  which  will  always  retain  some  color. 

In  the  meantime  heat  to  boiling  about  icx)  volumes 
of  distilled  water  in  a  clean  enameled  container.  When 
the  tissue  has  been  completely  freed  of  blood,  add  5 
drops  of  acetic  acid  for  each  liter  of  water  and  imme- 
diately throw  in  the  entire  mass  of  tissue.  Boil  steadily 
for  ten  minutes,  pour  the  entire  contents  into  a  sieve, 
and  bring  another  measure  of  water  (from  now  on  with- 
out adding  acid)  to  boiling.  In  the  meantime  wash  the 
tissue  on  the  sieve  for  about  five  minutes  in  distilled 
water.  When  the  water  is  again  boiling,  throw  the 
tissue  into  it  again,  and  again  boil  for  ten  minutes. 
Repeat  this  process  five  or  six  times.  After  the  last 
boiling,  heat  only  about  5  volumes  of  water  instead  of 
100  and  throw  the  tissue  into  it.  Boil  for  five  minutes, 
carefully  avoiding  scorching.  Filter  off  some  of  the 
cooking  water  through  a  hard  filter-paper.  To  5  c.c. 
of  the  filtrate  add  at  least  i  c.c.  of  i  per  cent,  aqueous 
ninhydrin  solution,  as  described  below,  and  boil  in  the 
flame  for  one  minute.  If  absolutely  no  violet  color  is 
visible  after  standing  half  an  hour,  and  provided  the 
tissue  remains  perfectly  white,  the  preparation  is  com- 
plete. Otherwise  the  boiling  for  five  minutes  in  5  volumes 
of  water,  alternating  with  five  minutes'  washing  in  cold 
water,  is  repeated  until  the  desired  result  is  obtained. 
Then  place  in  a  sterile,  wide-mouthed,  glass-stoppered 
bottle,  add  a  little  sterile  water,  and  a  good  deal  of  chloro- 
form and  toluol.  A  properly  prepared  organ  will  keep 
indefinitely  if  it  does  not  become  infected.  Remove 
portions  only  with  sterile  forceps,  and  never  put  back  a 


534  SERODIAGNOSTIC  METHODS 

portion  once  removed.  The  layer  of  toluol  must  touch 
the  stopper,  and  bits  of  tissue  must  not  extend  above  the 
toluol. 

Before  used  for  testing  unknown  sera  the  substrate 
must  be  shown  to  react  negatively  with  a  variety  of 
known  negative  sera,  and  positively  with  known  positive 
sera. 

(2)  Dialyzing  Membrane. — The  dialyzing  shells  No. 
579A  of  Schleicher  and  Schiill  are  employed.  Before 
use  they  must  be  shown  to  be  impermeable  to  albumin 
and  all  equally  permeable  to  silk  peptone.  Shells  already 
proved  can  be  purchased  from  Schops,  Halle  an  der 
Salle,  but  these  must  be  retested  before  use,  and  again 
from  time  to  time,  to  avoid  error.  These  tests  are  made 
as  follows: 

(a)  To  Show  Impermeability  to  Albumin. — Place  5  c.c. 
of  perfectly  fresh  egg-white,  from  which  the  membranes 
and  any  floccules  have  been  removed,  in  a  cylinder 
graduate,  and  add  95  c.c.  of  water.  Mix  by  vigorous 
shaking.  In  the  meantime  soak  the  shells  about  one- 
half  hour  in  cold  water.  Place  each  shell  in  a  clean 
loo-c.c.  Ehrlenmeyer  flask,  and  add  to  each  2.5  c.c.  of 
the  albumin  solution.  Use  a  graduated  pipet  for  this 
purpose,  introducing  well  into  the  mouth  of  the  shell, 
and  carefully  avoiding  touching  the  outside  of  the  shell 
with  the  pipet.  Close  the  upper  end  of  the  shell  between 
finger  and  thumb,  and  rinse  the  outside  of  the  shell 
thoroughly  in  running  water.  Then  close  the  shell  by 
pinching  near  its  center,  and  rinse  the  upper  end,  inside 
and  outside,  in  the  same  way.  The  hands  should  be 
thoroughly    clean    and    recently    washed,    or    perhaps 


THE  PROTECTIVE   FERMENT   REACTIONS  535 

covered  by  clean  sterile  gloves.  Still  better,  the  shells 
should  be  handled  only  with  clean  forceps. 

Now  place  each  shell  in  a  fresh  Ehrlenmeyer  flask,  or 
large  test-tube  with  a  foot,  containing  20  c.c.  of  sterile 
distilled  water.  Cover  both  the  water  and  the  contents 
of  the  shell  about  |  cm.  deep  with  toluol,  taking  care 
that  the  level  of  fluid  in  both  shall  be  the  same.  Cover 
the  flask  or  test-tube  with  an  inverted  watch-glass  and 
place  in  the  incubator  for  about  sixteen  hours. 

Arrange  the  flasks  in  order  on  the  work  table,  and  with 
a  pipet,  closed  with  the  finger  while  it  is  quickly  passed 
through  the  layer  of  toluol,  transfer  10  c.c.  of  the  fluid 
on  the  outside  of  the  shell  to  a  test-tube.  A  separate, 
absolutely  clean  and  dry  pipet  must  be  used  for  each 
transfer.  Add  to  each  test-tube  2.5  c.c.  of  2,7,  per  cent, 
caustic  soda  solution,  and  mix  by  careful  shaking,  but 
do  not  use  the  thumb  as  a  stopper.  The  skin  excretions 
may  contain  substances  which  will  cause  a  reaction. 
Add  to  each  tube  about  i  c.c.  of  copper  sulphate,  about 
0.2  per  cent.,  so  as  to  form  a  layer  on  the  surface. 
Examine  the  line  of  contact  by  transmitted  light.  The 
slightest  trace  of  violet  or  rose  color  shows  that  the 
shell  is  permeable  to  albumin  and  it  must  be  discarded. 
To  be  on  the  safe  side,  discard  any  about  which  there  is 
any  doubt. 

The  ninhydrin  reaction  is  less  suited  for  determining 
permeability  to  albumin  than  the  biuret  reaction, 
because  the  former  reacts  strongly  with  digested  protein, 
but  only  weakly  with  undigested  protein.  It  must  be 
remembered,  however,  that  the  ninhydrin  reaction 
depends  on  the  concentration  of  the  decomposition 
products,  and  that  it  is  additive.    A  reaction  may  occur 


536  SERODIAGNOSTIC  METHODS 

when  concentrations  of  albumin  and  peptone,  neither 
of  which  reacts  alone,  are  brought  together.  Individ- 
uals unable  to  recognize  slight  traces  of  the  biuret  reac- 
tion must  use  some  other  test  for  the  albumin.  One 
may  make  use  of  a  homologous  immune  serum  of  very 
high  titer,  as  described  above  for  determining  meat 
adulteration,  or  may  use  the  ninhydrin  test,  first  having 
determined  the  maximum  amount  of  albumin  which 
can  be  added  to  known  impermeable  shells  without 
obtaining  a  positive  reaction. 

(b)  To  Show  Uniform  Permeability  to  Silk  Peptone. — 
Empty  the  shells  not  discarded  as  a  result  of  the  first 
test,  place  them  in  a  sieve  and  wash  in  flowing  water 
for  half  an  hour,  and  place  in  boiling  water  for  not  more 
than  half  a  minute.  Boiling  injures  the  shells,  rendering 
them  thicker  and  less  permeable.  Handle  the  shells 
only  with  forceps  sterilized  in  the  flame.  Arrange  in 
flasks  as  before,  and  add  to  each,  using  the  same  precau- 
tions, 1.5  c.c.  of  a  I  per  cent,  solution  of  silk  peptone 
(Hochst).  Wash  as  before  and  place  in  flasks  or  tubes 
containing  20  c.c.  of  sterile  distilled  water.  Cover  with 
toluol,  close  the  flask,  and  incubate  for  about  sixteen 
hours.     Evaporation  must  be  prevented. 

Remove  10  c.c.  of  the  dialysate  as  before,  place  in  large 
test-tubes  (10  by  i  inches),  and  add  to  each  tube  0.2 
c.c.  of  ninhydrin  solution  prepared  as  follows:  The  nin- 
hydrin is  packed  in  vials  containing  o.i  gm.  Empty  a 
vial  into  a  lo-c.c.  volumetric  flask.  Rinse  the  vial  sev- 
eral times  with  sterile  distilled  water,  adding  rinsings  to 
the  flask.  Fill  the  flask  nearly  full  and  place  in  the  incu- 
bator till  dissolved.    Cool  and  fill  to  the  mark. 

The  solution  is  affected  by  light  and  does  not  keep  well, 


THE  PROTECTIVE   FERMENT   REACTIONS  537 

hence  only  small  amounts  should  be  prepared  at  one 
time. 

Now  place  in  each  test-tube  a  glass  rod  about  lo  cm. 
long  by  4  mm.  in  diameter,  previously  cleaned  and  dried 
in  the  dry  air  sterilizer,  and  kept  in  a  glass-stoppered 
bottle.  They  must  be  handled  only  with  forceps.  The 
rod  prevents  "bumping,"  and  insures  uniform  evapora- 
tion during  the  next  step.  This  step  is  almost  the  most 
important  in  the  entire  procedure.  By  means  of  a  holder, 
place  each  tube  in  succession  in  the  center  of  a  high 
Bunsen  flame;  as  soon  as  bubbles  begin  to  form,  move  to 
the  edge  of  the  flame.  Keep  the  tube  boiling  vigorously 
(halfway  up  the  tube)  for  exactly  one  minute  from  the 
time  bubbles  begin  to  form.  Since  the  intensity  of  the 
reaction  depends  on  the  concentration  of  the  fluid  and 
the  amount  of  heat  applied,  it  is  of  vital  importance  that 
the  boiling  of  all  tubes  shall  be  identical.  As  soon  as 
the  heating  is  complete,  make  sure  that  all  tubes  contain 
the  same  amount  of  fluid.  This  is  made  easier  by  mark- 
ing the  lo-c.c.  level  on  each  tube  before  it  is  used.  Allow 
all  the  tubes  to  stand  for  half  an  hour.  A  certain  depth 
of  color  will  be  found 'to  predominate.  Discard  all  the 
shells  the  dialysate  of  which  jdelds  either  a  lighter  or 
darker  color  than  this  standard.  Again  wash  and  boil 
the  shells  as  above,  place  in  a  sterile  flask,  add  sterile 
distilled  water  and  equal  volumes  of  toluol  and  chloro- 
form, so  as  to  fill  the  flask  entirely  full.  They  are  now 
ready  for  use.  Remove  from  the  flask  only  with  sterile 
forceps. 

(3)  The  patient's  blood  must  be  drawn  while  fasting, 
i.  e.,  before  breakfast.  The  senmi  must  be  devoid  of 
formed  elements,   and  must  show  no  hemoglobin  on 


538  SERODIAGNOSTIC   METHODS 

spectroscopic  examination.  Puncture  the  vein  with  a 
sterile  and  absolutely  dry  needle,  and  receive  15  to  20 
c.c.  of  blood  in  a  clean,  dry,  sterile  tube.  After  the  serum 
has  separated  spontaneously  (five  or  six  hours),  transfer 
the  serum  to  a  centrifuge  tube  (also  clean,  dry,  and  ster- 
ile) and  centrifugate  live  to  ten  minutes.  Serum  cannot 
be  used  when  more  than  twelve  hours  old,  and  must  be 
sterile. 

2.  The  Test  Proper. — Abderhalden  lays  down  the 
following  fundamental  laws  to  be  observed: 

(i)  Absolute  cleanliness  of  room,  work-bench,  all 
glassware,  and  utensils.     The  latter  must  also  be  dry. 

(2)  Only  freshly  distilled  sterile  water  must  be  used. 
All  work  is  to  be  done  on  an  aseptic  and  antiseptic  basis. 

(3)  No  bacteriologic  or  chemical  work  may  be  done 
in  the  room.  The  incubator,  especially,  must  be  used 
for  no  other  purpose. 

(4)  Good  light  must  be  had.  Artificial  light  should 
never  be  used.  Not  more  than  five  or  six  tests  should 
be  attempted  at  one  time. 

(5)  The  worker  must  understand  not  only  the  method, 
but  the  underlying  principles. 

While  the  serum  is  separating,  prepare  and  arrange  all 
the  apparatus.  Remove  with  sterile  forceps  the  amount 
of  substrate  needed.  Cover  with  5  volumes  of  water 
and  boil  for  five  minutes.  Test  5  c.c.  of  the  filtrate 
with  i.o  c.c.  of  I  per  cent. ninhydrin,  as  described  above. 
The  substrate  may  be  used  only  when  not  a  trace  of  color 
is  visible  after  boiling  one  minute  and  standing  thirty 
minutes. 

Place  as  many  proved  shells  as  will  be  required  in 
clean,  dry  Ehrlenmeyer  flasks  and  add  to  each  about 


THE   PROTECTIVE   FERMENT  REACTIONS  539 

0.5  gm.  of  the  substrate,  crushing  and  tearing  the  latter 
with  forceps  to  increase  its  surface  area.  One  shell  for 
each  serum  to  be  tested  should  be  left  without  sub- 
strate. To  each  shell  add  about  1.5  c.c.  of  serum. 
The  same  amount  of  sermn  should  be  placed  in  aU. 
Wash  the  shells  as  already  described  (see  p.  534)  and 
transfer  to  fresh  flasks  or  tubes  containing  20  c.c.  sterile 
distilled  water.  Pour  a  large  amount  of  toluol  in  each 
shell  and  over  the  water  outside,  and  thoroughly  wet 
the  part  of  the  shell  protruding  above  the  fluid  with 
toluol.  Place  in  incubator  at  37°  C.  for  sixteen  hours. 
On  removing,  toluol  must  still  be  present  inside  and 
outside  the  shell.  Arrange  the  (numbered)  flasks  with- 
out special  order  on  the  work-bench.  Remove  10  c.c.  of 
the  dialysate  from  each  in  the  manner  already  described. 
Place  in  large,  clean,  dry  test-tubes.  Add  to  each  0.2 
c.c.  of  the  ninhydrin  solution,  and  a  clean  dry  bumping 
rod,  and  boil  one  after  another  as  above  described. 
Allow  all  tubes  to  stand  for  one-half  hour.  Now  de- 
termine which  tubes  show  color,  and  then  determine  the 
proper  place  of  each  in  the  series. 

If  serum  alone  gives  no  color,  while  senun  plus  sub- 
strate shows  color,  even  the  slightest,  the  test  is  positive, 
provided  heating  and  evaporation  have  been  exactly  alike 
for  both.  When  the  dialysate  of  the  serum  alone  shows 
color  (due,  for  example,  to  the  presence  of  hemoglobin  or 
products  of  digestion  in  the  senun),  the  reaction  may  be 
regarded  as  a  vaUd  positive  reaction,  provided  the  serum 
plus  substrate  shows  a  distinctly  darker  color. 

The  biuret  reaction  may  be  used  in  place  of,  or  with, 
the  ninhydrin  reaction  by  any  worker  who  is  able  to 
recognize  the  slightest  trace  of  this  reaction. 


54©  SERODIAGNOSTIC  METHODS 

B.  The  Polariscopic  Method 
I.  Preparation  of  the  Organ  Peptone. — The  tissue  is_ 
freed  of  blood  and,  if  necessary,  of  fats  and  lipoids  in 
the  manner  already  described  (see  p.  532).  Remove 
water,  as  far  as  possible,  by  pressing  between  sheets  of 
absorbent  paper.  Then  place  the  tissue  in  cold  70  per 
cent,  sulphuric  acid  (volume  per  cent.).  Shake  vigor- 
ously and  cork  well.  Repeat  the  shaking  from  time  to 
time.  The  tissue  dissolves  shortly,  and  the  solution 
takes  on  a  brownish  color.  The  temperature  must  not 
be  allowed  to  rise  above  20°  C.  At  the  end  of  exactly 
three  days  place  the  bottle  containing  the  solution  in 
ice- water,  and  add  slowly  10  volumes  of  distilled  water, 
taking  care  that  the  temperature  does  not  rise  above 
20°  C.  Now  add  slowly  baryta  in  substance,  still  keep- 
ing the  temperature  down,  until  the  filtrate  (or  centrif- 
ugate)  gives  a  precipitate  with  neither  dilute  baryta 
water  nor  dilute  sulphuric  acid.  Approximately  105 
gm.  of  the  soHd  baryta  neutralize  100  c.c.  of  the  70  per 
cent,  sulphuric  acid.  The  first  stages  of  the  neutraliza- 
tion can  be  controlled  with  litmus-paper.  As  the  end- 
point  is  approached,  a  small  amount  is  filtered  or  cen- 
trifugated,  as  above  indicated,  and  portions  tested  for 
excess  of  acid  and  base  respectively  by  adding  very  dilute 
solutions  of  the  base  or  acid  to  each.  A  precipitate  on 
adding  baryta  water,  which  dissolves  on  heating  gently 
with  nitric  acid,  is  a  baryta  salt  of  peptone,  and  calls 
for  the  addition  of  more  baryta. 

When  no  precipitate  is  obtained,  filter  through  a 
double-plaited  filter,  or  under  negative  pressure  through 
a  hard  paper,  with  animal  charcoal.  Centrifugation  is 
more  rapid  and  may  be  used.    For  economy's  sake,  wash 


THE  PROTECTIVE   FERMENT  REACTIONS 


541 


the  precipitate  several  times  with  distilled  water  and  add 
the  wash-water  to  the  solution.  The  ninhydrin  test 
can  be  employed  to  determine  when  all  the  peptone  has 
been  recovered  from  the  precipitate. 

In  the  meantime  the  evaporation  of  the  solution  under 
negative  pressure  is  begun.    A  convenient  apparatus  is 


To  Drain 


Fig.  176. — Abderhalden's  apparatus  for  distilling  under  negative  pressure  at  low  temper- 
ature. 

figured  by  Abderhalden,  and  reproduced  here  (Fig.  176). 
It  permits  the  evaporation  of  the  water  under  strong 
negative  pressure  at  a  temperature  not  to  exceed  40°  C. 
The  separatory  funnel  (2)  refills  automatically  from  the 
reservoir  (i).   The  solution,  which  is  allowed  to  enter  the 


542  SERODI AGNOSTIC   METHODS 

distilling  flask  (3)  immersed  in  hot  water  (4)  drop  by  drop, 
vaporizes  at  once,  the  vapor  passing  through  the  side 
arm,  where  it  condenses  in  the  second  flask  (6),  over 
which  flows  a  stream  of  cold  water  to  be  collected  in  the 
funnel  (7).  The  flask  is  supported  in  the  funnel  by 
hooks.  The  negative  pressure  is  furnished  by  the  Chap- 
man pump.  High  temperatures  must  be  avoided 
throughout,  unless  one  is  certain  that  neither  free  acid 
nor  free  baryta  is  present.  As  the  concentration  in- 
creases, any  trace  of  either  present  in  the  original  solu- 
tion is  also  concentrated,  and  may  cause  further  hydro- 
lysis of  the  peptone  if  the  temperature  is  permitted  to 
rise. 

The  final  residue  is  bright  yellow  and  syrupy.  Mix 
with  100  volumes  of  methyl  alcohol,  boil,  and  filter  hot 
through  a  plaited  filter  into  about  5  volumes  of  ethyl 
alcohol.  Place  in  ice-water.  The  addition  of  ether 
renders  the  precipitation  more  complete.  Begin  filtra- 
tion as  soon  as  the  precipitate  begins  to  grow  flocculent. 
Do  not  permit  the  filter  to  become  empty  till  the  last 
funnelful  has  been  added.  Place  the  filter-paper  with 
its  contents  in  a  vacuum  desiccator.  Drying  is  complete 
in  two  or  three  days.  It  may  now  be  collected  and 
weighed.  Dissolve  a  small  amount  in  enough  0.9  per 
cent,  salt  solution  to  make  a  10  per  cent,  solution,  and 
determine  its  rotating  power  at  37°  C.  with  the  polar- 
iscope.  If  the  rotation  is  more  than  i  degree,  dilute 
with  salt  solution  till  it  is  reduced  to  about  0.75  degree. 
This  is  merely  for  the  sake  of  economy  of  material. 

2.  Proving  the  Peptone  Solution. — Assuming  that  we 
are  dealing  with  placental  peptone,  place  in  a  test-tube 
I  c.c.  of  the  undiluted  serum  of  a  non-pregnant  individual 


THE   PROTECTIVE   FERMENT   REACTIONS  543 

(a  male).  The  serum  must  conform  to  the  requirements 
laid  down  for  serum  to  be  used  in  the  dialyzation  method. 
Add  I  c.c.  of  the  peptone  solution,  mix,  and  place  in  a 
2-c.c.  polariscope  tube.  Place  in  the  incubator  till  the 
temperature  reaches  37°  C.  Determine  the  rotating 
power  at  hourly  intervals.  If  there  is  no  change,  the 
peptone  certainly  contains  free  acid,  or  free  baryta,  as 
the  case  may  be.  If  there  is  a  change,  make  readings 
at  hourly  intervals,  to  establish  the  normal  curve,  by 
entering  the  readings  on  a  chart  in  which  the  abscissa 
corresponds  to  the  readings,  and  the  ordinate  corres- 
ponds to  the  intervals  between  readings.  The  normal 
curve  once  established  is  compared  with  readings  taken 
in  the  actual  test  only  at  four-  to  six-hour  intervals. 
Placental  peptone  is  now  on  the  market.  Other  organ 
peptones  must  be  prepared  by  the  worker. 

The  individual  worker  must  establish  by  trying  the 
margin  of  error  for  successive  readings  of  the  same  fluid 
in  his  own  case.  This  is  usually  about  0.02  degree. 
For  the  test  this  is  doubled.  A  change  of  0.5  degree  or 
more  is  required  to  constitute  a  positive  reaction. 

3.  The  Test  Proper. — This  is  carried  out  in  essentially 
the  same  manner  as  the  testing  of  the  peptone  solution. 
The  work  must,  of  course,  be  done  aseptically.  The 
serum  used  must  conform  to  the  requirements  laid 
down  for  serum  to  be  used  in  the  dialyzation  method. 
In  each  of  three  2-c.c.  polariscope  tubes  place  respec- 
tively I  c.c.  peptone  solution  plus  i  c.c.  salt  solution; 
I  c.c.  patient's  serum  plus  i  c.c.  salt  solution;  and  i 
-c.c.  peptone  solution  plus  i  c.c.  patient's  serum.  Place 
all  tubes  in  the  incubator  or  water-bath  at  37°  C,  and 
make  readings  at  four-  to  six-hour  intervals  up  to  about 


544  SERODIAGNOSTIC  METHODS 

sixteen  hours.  Unless  the  polariscope  is  provided  with 
some  adequate  means  for  maintaining  a  constant  tem- 
perature, the  tubes  must  be  removed  from  the  incu- 
bator, placed  in  the  polariscope,  and  read  in  the 
shortest  possible  time.  When  the  reading  in  the  third 
tube,  containing  both  peptone  and  serum,  varies  by 
0.05  degree  or  more  from  that  given  by  the  other  tubes, 
the  reaction  is  positive. 

The  only  sources  of  error  in  the  method  are:  (a)  Pre- 
cipitates or  cloudiness  of  the  solution.  This  is  rare, 
and  excludes  error  by  rendering  any  reading  whatsoever 
impossible,  {h)  Errors  arising  from  making  the  readings 
at  other  than  a  uniform  temperature.  The  first  reading 
should  be  made  at  the  end  of  one  hour;  the  second, 
two  hours  later,  and  subsequent  readings  at  4-  to  6-hour 
intervals.  No  reading  should  be  attempted  after  thirty- 
six  to  forty-eight  hours,  {c)  Most  important,  errors 
arising  in  the  observer,  due  to  visual  fatigue.  Only  a 
few  readings  should  be  attempted  at  the  same  time,  and 
not  more  than  thirty  seconds  should  be  given  to  making 
a  single  reading. 


APPENDIX 


L  APPARATUS,  REAGENTS,  AND  STAINS 

The  apparatus  and  reagents  listed  here  are  sufficient 
for  all  but  the  rarer  tests  described  in  the  text.  Those  in 
smaller  type  are  less  frequently  required.  For  ordinary 
routine  work  a  much  smaller  hst  will  suffice. 

A.  APPARATUS 

Beakers  and  flasks,  several  sizes,  preferably  of  Jena 
glass. 

Blood  lancet,  or  some  substitute  (see  Fig.  67). 

Bunsen-burner  or  alcohol  lamp. 

Buret,  25-c.c.  capacity,  preferably  with  Schellbach 
stripe. 

Buret  and  filter-stand  combined. 

Centrifuge — hand,  electric,  or  water-power  (see  Figs. 
20,  21).  With  the  last  two  a  speed  indicator  is  desirable. 
Radius  of  arm  when  in  motion  should  be  6f  inches. 
Plain  and  graduated  tubes  accompany  the  instrument; 
milk- tubes  (see  Fig.  165)  must  be  purchased  separately. 
The  hematocrit  attachment  (see  Fig.  81)  is  not  much 
used. 

Corks,  preferably  of  rubber,  with  one  and  two  holes. 

Cover-glasses,  No.  i  or  No.  2  thickness — f-inch 
squares  are  most  convenient. 

Cover-glass  forceps,  preferably  Stewart's  (see  Fig. 
87). 

35  545 


546  APPENDIX 

Esbach's  tube  (see  Fig.  29). 

Evaporating  dish. 

Filter-paper:  ordinary  cheap  paper  for  urine  filtra- 
tion; "ashless"  quantitative  filter-paper  for  chemical 
analyses. 

Glass  funnels. 

Glass  rods  and  tubing  of  sodium  glass:  for  stirring  rods, 
urinary  pipets,  etc. 

Glass  slides:  the  standard  i-  by  3-inch  size  will  answer 
for  all  work,  although  a  few  larger  slides  will  be  found 
convenient;  those  of  medium  thickness  are  preferable. 

Graduates,  cylindric  form,  several  sizes.  At  least 
one  of  these  (the  50  c.c.  is  most  useful)  should  be 
certified. 

Graniteware  basin. 

Hemoglobinometer :  see  pp.  226  to  232  for  descriptions 
of  the  different  instruments. 

Hemacytometer :  either  Tiirck,  Neubauer,  or  Zappert 
ruHng  is  desirable  (see  Figs.  75,  76,  82). 

Hypodermic  syringe:  the  "Aseptic  Sub-Q,  Tubercu- 
lin," is  probably  the  most  useful  type. 

Incubator  (see  p.  455). 

Labels  for  slides  and  bottles. 

Litmus-paper,  red  and  blue,  Squibb's  preferred. 

Mett's  tubes  (see  p.  339). 

Microscope  (see  Fig.  i).  Equipment  described  on 
p.  42. 

Petri  dishes. 

Platinum  wires  (see  p.  455). 

Sterilizers:  the  Arnold  type  for  steaming;  oven  lor  dry 
sterilization  (see  p.  454). 

Stomach-tube. 


REAGENTS   AND   STAINS  547 

Test-glass,  conical,  one  side  painted  half-white,  half- 
black. 
Test-tubes,  rack,  and  cleaning  brush. 
Ureometer,  Doremus-Hinds'  pattern  (see  Fig.  24)! 
Urinometer,  preferably  Squibb's  (see  Fig.  17). 

Blood-fixing  oven,  or  Kowarsky's  plate  (see  Fig.  88). 

Copper-foil  and  gauze. 

Cotton,  absorbent,  for  filtering,  etc. 

"Cotton-batting"  for  plugging  tubes. 

Culture-media.  The  selection  depends  upon  the  work 
to  be  done  (see  p.  459). 

Holt's  cream  gage  and  hydrometer  (see  Fig.  164). 

Horismascope  (see  Fig.  28). 

Pipets,  graduated,  5-  to  50-c.c.  capacity.  For  some  sero- 
diagnostic  methods  a  few  pipets  of  lo-c.c.  capacity  gradu- 
ated in  one-tenths,  and  o.i-c.c.  graduated  in  one-one- 
hundredths,  will  be  required. 

Ruhemann's  tube  for  uric-acid  estimation  (see  Fig.  27). 

Saccharimeter  (see  Fig.  31). 

Scales,  about  o.i  to  100  gm.  capacity. 

Strauss'  separatory  fimnel  for  lactic-acid  test  (see  Fig.  106). 

Suction  filter. 

Urinopyknometer  of  Saxe  (see  Fig.  18). 

Widal  reaction  outfit:  either  living  agar  cultures  of  the 
typhoid  bacillus,  or  the  dead  cultures  with  diluting  apparatus, 
which  are  sold  under  various  trade  names. 

Water-bath. 

B.  REAGENTS  AND  STAINS 

All  stains  and  many  reagents  are  best  kept  in  small 

.dropping  bottles,  of  which  the  flat  top  "T.  K."  pattern 

is  probably  most  satisfactory.     Formulae  are  given  in 

the  text.     Dry  stains  (Grubler's  should  be  specified) 


548  APPENDIX 

and  most  staining  solutions  and  chemical  reagents  can 
be  purchased  of  the  Denver  Fire  Clay  Co.,  Denver, 
Colorado;  Bausch  &  Lomb  Optical  Co.,  Rochester, 
New  York;  or  Eimer  &  Amend,  New  York.  For  the 
physician  who  does  only  a  small  amount  of  work  the 
''Soloid"  tablets  manufactured  by  Burroughs,  Well- 
come &  Co.  are  convenient  and  satisfactory.  These 
tablets  have  only  to  be  dissolved  in  a  specified  amount 
of  fluid  to  produce  the  finished  stain.  Most  of  the 
stains  and  many  of  the  reagents  mentioned  here  come 
in  this  form. 

Acid,  glacial  acetic.  Other  strengths  can  be  made 
from  this  as  desired. 

Acid,  hydrochloric,  concentrated  (contains  about  32 
per  cent,  by  weight  of  absolute  hydrochloric  add). 
Other  strengths  can  be  made  as  desired. 

Acid,  nitric,  strong,  colorless. 

Acid,  nitric,  yellow.  Can  be  made  from  colorless  acid 
by  adding  a  splinter  of  pine,  or  allowing  to  stand  in 
sunlight. 

Acid,  sulphuric,  concentrated. 

Alcohol,  ethyl  (grain-alcohol) .  This  is  ordinarily  about 
93  to  95  per  cent.,  and  other  strengths  can  be  made  as 
desired. 

Aqua  ammoniae  fortior  (sp.  gr.  0.9). 

Bromin,  or  Rice's  solutions  (see  p.  1 18),  for  urea  estima- 
tion. 

Chloroform. 

Diluting  fluid  for  erythrocyte  count  (see  p.  240). 

Diluting  fluid  for  leukocyte  count  (see  p.  258). 

Dimethyl-amido-azobenzol,  0.5  per  cent,  alcoholic 
solution. 


REAGENTS   AND   STAINS  549 

Distilled  water. 

Esbach's  or  Tsuchiya's  reagent  (see  p.  134). 
Ether,  sulphuric. 

Ferric  chlorid:  saturated  aqueous  solution  and  10  per 
cent,  aqueous  solution. 
Guaiac,  powdered. 
Haines'  (or  Fehling's  or  Benedict's)  solution  (see  pp. 

138,  139)- 

Lugol's  solution  {Liquor  lodi  Compositus,  U.  S.  P.). 
Gram's  iodin  solution  (see  p.  74)  can  be  made  from  this  by 
adding  fourteen  times  its  volume  of  water. 

Obermayer's  reagent  (see  p.  114). 

Oil  of  cedar  for  immersion. 

Phenylhydrazin,  pure. 

Phenol. 

Phenolphthalein,  i  or  0.5  per  cent,  alcoholic  solution. 

Purdy's  (or  Fehling's  or  Benedict's)  solution  (see  pp. 
142-144). 

Robert's  reagent  (see  p.  132). 

Sodium  chlorid  (table  salt),  saturated  aqueous  solu- 
tion. 

Sodium  hydroxid  (caustic  soda),  40  per  cent,  solution; 
other  strengths  can  be  made  from  this  as  desired. 

Sodium  hydroxid,  decinormal  solution.  The  prac- 
titioner will  find  it  best  to  purchase  this  solution  ready 
prepared.  Elmer  and  Amend,  New  York,  and  many 
other  chemical  supply  houses  carry  it  in  stock.  For 
ordinary  clinical  work  41  gm.  of  Merck's  "sodium 
hydrate  by  alcohol"  from  a  freshly  of>ened  bottle  may 
be  dissolved  in  1000  c.c.  water.  This  makes  a  normal 
solution  and  must  be  diluted  with  9  volumes  of  water  to 
make  the  decinormal  solution. 


55©  APPENDIX 

Sodium  nitrite,  0.5  per  cent,  solution  for  diazo-reaction. 
Must  be  freshly  prepared. 

Sulphanilic  acid  solution  for  diazo-reaction   (see  p. 
161). 

■  Stains. — It  will  be  most  satisfactory  to  have  on  hand  a 
stock  of  dry  stains  (which  keep  well)  and  to  make  the  solu- 
tions as  needed.  Ordinarily  the  smallest  quantity  ob- 
tainable in  an  unbroken  package  should  be  purchased. 
The  following  dry  stains  should  be  sufficient  for  the 
ordinary  clinical  laboratory:  Eosin,  w.  g.;  fuchsin,  acid; 
fuchsin,  basic;  gentian- violet;  methylene-blue,  B.  X.  or 
Ehrlich's  rectified;  methyl-green;  pyronin;  Sudan  III; 
Wright's  stain.  The  most  frequently  used  solutions 
are: 

Carbolfuchsin  (see  p.  65). 

Eosin,  saturated  aqueous  solution. 

Formalin-gentian-violet,     or     anilin-gentian-violet 
(seep.  74). 

Gabbet's    stain    or   Pappenheim's   methylene-blue 
stain  (see  pp.  65,  66). 

Loffler's  alkaline  methylene-blue  solution   (see  p. 

74)- 
Pappenheim's  pyronin-methyl-green  stain   (see  p. 

467). 
Stain  for  fat:  Sudan  III,  saturated  solution  in  70 
per  cent,  alcohol;  or  i  per  cent,  aqueous  solution 
osmic  acid. 
Wright's,  Harlow's,  or  Jenner's  stain  for  blood. 
Tincture  of  guaiac,  diluted  to  a  light  sherry-wine  color 
(keep  in  a  dark  glass  bottle). 
Turpentine,  "ozonized"  (see  p.  157). 


REAGENTS   AND   STAINS  55 1 

Acid,  boric,  for  preserving  urine  (see  p.  86). 

Acid,  oxalic. 

Acid,  salicylous  (salicyl  aldehyd),  10  per  cent,  alcoholic 
solution. 

Alcohol,  amylic. 

Alcohol,  ethyl,  absolute. 

Alcohol,  methyl  (pure). 

Ammonium  sulphate,  C.  P. 

Antiformin  (see  p.  68). 

Barium  chlorid  mixture  (see  p.  112). 

Benzidin. 

Boas'  reagent  or  Giinzburg's  (see  p.  326). 

Benzol. 

Boggs'  reagent  (see  p.  444). 

Calcium  chlorid,  i  per  cent,  solution. 

Canada-balsam  in  xylol:  necessary  only  when  permanent 
microscopic  preparations  are  made. 

Carbon  disulphid. 

Charcoal,  animal. 

Chromium  trioxid. 

Congo-red,  strong  alcoholic  solution. 

Copper  sulphate. 

Diluting  fluid  for  blood-platelet  count  (see  pp.  260,  261). 

Egg-albumen  disks  in  glycerin  (see  p.  293). 

Ether,  acetic,  pure. 

Florence's  reagent  (see  p.  450). 

Formalin  (40  per  cent,  solution  of  formaldehyd  gas). 

Hydrogen  dioxid. 

India-ink  ('nach  Burri/'  Griibler)  (see  p.  448). 

lodin  crystals. 

Iron  sulphid. 

Lead  acetate  (sugar  of  lead) ;  used  in  10  per  cent,  solution 
•  to  clarify  urine. 

Lead  acetate,  tribasic 

Lime-water.  LlUl\M;V    CI 

cuLLizGiE  or  Ob'TLUlVaUl 


552  APPENDIX 

MuUer's  fluid  saturated  with  mercuric  chlorid  (see  p.  72). 
Orcinol. 

Pepsin,  U.  S.  P. 
Phenylhydrazin  hydrochlorid. 
Potassium  ferrocyanid,  10  per  cent,  solution. 
Potsssium  oxalate  (neutral). 
Potassium  persulphate. 
Resorcinol. 

Ruhemann's  reagent  (see  p.  125). 

Silver  nitrate  crystals;  also  12  per  cent,  aqueous  solu- 
tion, and  "ammoniated"  solution  (see  p.  123). 
Sodium  alizarin  sulphonate,  i  per  cent,  aqueous  solution. 
Sodium  carbonate. 

Sodium  chlorid,  2  per  cent,  solution;  from  this  normal  salt 
solution  (0.8  per  cent.)  can  be  made  as  desired. 
Sodiiun  hyposulphite. 
Sodium  nitroprussid. 
Sodiimi  sulphate. 
Staining  solutions: 

Carbol-thionin. 

Ehrlich's  triple  stain  for  blood. 

Fuchsin,  weak  solution;  can  be  made  when  desired  by 
adding  a  little  carbolfuchsin   to  a  test-tube  of  water. 

Gentian-violet,  saturated  alcoholic  solution. 

Giemsa's  stain  (see  p.  447). 

Methylene-blue  and  borax  solution  (see  p.  302). 

Methylene-blue,  saturated  aqueous  solution  for  blood. 

Van  Gieson's  stain  for  Negri  bodies  (see  p.  452). 
Sulphiu',  powdered. 

Talc,  purified  (Talcum  Purificatimi,  U.  S.  P.). 
Toluol. 

Trichloracetic  acid  solution  (see  p.  131). 
Uranium  nitrate,  5  per  cent,  aqueous  solution. 
Xylol. 
Zinc,  arsenic-free. 


WEIGHTS  AND  MEASURES 


553 


IL  WEIGHTS,   MEASURES,  ETC,  WITH 
EQUIVALENTS 


Meter  (unit  of  length) : 

Gram  (unit  of  weight) : 
Liter  (unit  of  capacity) : 


METRIC 

MilUmeter  (mm.)  =  j,^^  meter. 
Centimeter  (cm.)  =  ^J^  meter. 
Kilometer  =  looo  meters. 

Micron  (ji)  =  y^^j  millimeter. 

Milligram  (mg.)  =  jb»j5  gram. 
Kilogram  (kilo.)    =  looo  meters. 

Cubic  Centimeter  =:  joVg  liter.      Same  measure  as  milli- 
liter (ml.). 


I  Millimeter  = 
I  Centimeter  = 
X  Meter  = 

I  Micron  (ft)  = 


fo.03937  (jigapprox.)  in. 

1000  microns. 
to-3937(§approx.)in. 
I  0.0328  feet. 
I  39.37  in. 
[  3.28  feet. 

lo.ooi  millimeter. 


I  Gram.       = 

z  Kilogram  = 
s  Liter         = 


JS-43  grams. 

0.563  dram     ^ 

0.035  ounce     >  Avoir. 

0.0022  pound) 

0.257  dram      J 

0.032  ounce     >-Apoth. 

0.0027  pound  j 

35.27  ounce  (Avoir.). 

2.2  pound  (Avoir.). 

1.056  (i  approx.)  quart. 

61.02  cu.  inches. 

1000  cu.  centimeters. 


I  Sq.  Millimeter  =:  0.00155I 

I  Sq.  Centimeter  =  0.1550    >-sq.  in. 

1  Sq.  Meter  =  1550      J 

I  Sq.  Meter  =  10.76  sq.  feet. 

1  Inch         =  25.399  millimeters. 
1  Sq.  Inch  =  6.451  sq.  centimeters. 
I  Cu.  Inch  =  16.387  cu.  centimeters. 


I  Cu.  Millimeter  =  0.00006 )         . 
I  Cu.  Centimeter  =  0.0610  J  ' 

I  Cu.  Centimeter  =  o.ooi  liter. 

.  f  35-32  cu.  feet. 

'  (  61025.4  CU-  in. 


I  Cu.  Meter  =  ■ 


t  Foot         =  30.48  centimeters. 
I  Sq.  Foot  =  0.093  sq.  meter. 
X  Cu.  Foot  =  0.028  cu.  meter. 


AVOIRDUPOIS  WEIGHT 


,  Ounce  ={437^5^- 
X  Pound  =  16  ounces. 


I  Grain    =  0.065  (A  approx.) 
I  Dram   =  1.77  (ij  approx.) 
X  Ounce  =  28.35  (30  approx.) 
X  Pound  =  453-59  (5.°°  approx.) 
I  Pound  =  27.7  cu.  inches. 
X  Pound  =  1.215  Ih.  Troy. 


APOTHECARIES'   MEASURE 


I  Dram   =  60  minims. 
I  Ounce  =  8  drams. 
I  Pint      =  16  ounces. 
I  Gallon  =  8  pints. 


I  Dram   =3.70 

X  Ounce  =  29.57 

I  Pint      =  473.1 

X  Gallon  =  3785.4  . 

X  Gallon  =:  231  cu.  inches. 


cu.  centimeters. 


554 


APPENDIX 


APOTHECARIES'   WEIGHT 


I  Scruple  =  zo  grains. 
I  Dram    =  3  scruples  =  60  grains. 
I  Ounce   =  8  drams  =  480  grains. 
I  Pound  =  12  ounces. 


X  Grain  =  0.065 
I  Dram  =  3.887 
I  Ounce  =  31.10 
I  Pound  =  373.2 


To  convert 


mmtms 

jliiidounces 

grains 

drams 

cubic  centimeters 
cubic  centimeters 

grams 

grams 


into  cubic  centimeters  multiply  by   0.061 
"    cubic  centimeters         "  29.57 

"         "     0.0648 
"     3-887 


16.23 
0.0338 
IS-433 

o.as7 


grams 

minims 

jluidounces 

grains 

drams 


TEMPERATURE 


Centigrade. 


95 
90 

85 
80 

75 
70 
65 
60 

55 

50 

45 

44 

43 

42 

41 

40-5 

40 

39-5 

39 

38.5 

38 

37-5 


Fahrenheit. 

230° 
212 
203 
194 

185 
176 
167 
158 
149 
140 

13J: 
122 

113 
III. 2 
109.4 
107.6 
105.8 
104.9 
104 
103-1 
102.2 
101.3 
100.4 
99-5 


Centigrade. 


37° 

365 

36 

35-5 

35 

34 

Si 

32 

31 

3" 

25 

20 

15 
10 

+5 
o 

—5 
— 10 

—15 
— 20 


Fahrenheit. 

98.6° 

97-7 
96.8 

95-9 

95 

93-2 

91.4 

89.6 

87.8 

86 

77 
68 

59 

50 

41 

32 

23 

14 
+5 
—4 


0.54 
I 

2 

2-5 


1.8 
3-6 
4-5 


To  convert  Fahrenheit  into  Centigrade,  subtract  32  and 
multiply  by  0.555. 

To  convert  Centigrade  into  Fahrenheit,  multiply  by 
1.8  and  add  32. 


INDEX 


Abderhalden's  apparatus  for  dis- 
tilling under  negative  pressure, 

541 

protective  ferment  reaction,  530 
Absorption,  toxic,  degree  of,  283 
Absorptive  power  of  stomach,  344 
Accidental  albuminuria,  128 
Acetanilid  in  urine,  166 
Acetonuria,  149 

detection  of  acetone  in,  151 

Frommer's  test  in,  152 

Gunning's  test  in,  151 

Lange's  test  in,  152 

Legal's  test  for,  Lange's  modifi- 
cation, 152 

Lieben's  test  in.  Gunning's  modi- 
fication, 152 
Achard  and  Castaigne's  methylene- 

blue  test  for  urine,  98 
Achlorhydria,  335 
Acholic  feces,  350 
Achromatic  objectives,  24 
Achylia  gastrica,  stomach  contents 

in.  343 
Acid,  boric,  in  milk,  test  for,  445 
calcium  phosphate  in  urine,  185 
deficit  of,  in  gastric  contents,  337 
diacetic,  in  urine,  153 
hydrochloric,  combined,  319 

free,  319 
intoxication,  cause,  149 
lactic,  in  gastric  contents,  326 
oxybutyric,  in  urine,  154 
uric,   in   urine,    122.     See   also 

Uric  acid  in  urine. 
urine,  imorganized  sediments  in, 

176,  177 
urochloralic,  in  urine,  167 
Acid-fast  bacilli,  69 
Acidity  of  urine,  91 

quantitative  estimation,  92 


Acidity  of  urine,  quantitative  es- 
timation, Folin's  method,  92 
total,  of  gastric  contents,  333 
tests,  333 
Topfer's  test,  333 
Acidophilic  structures  of  blood,  266 
Acids,    free,    in   gastric    contents, 
tests  for,  325 
organic,  in  gastric  contents,  326 
quantitative  tests,  337 
Actinomyces  bovis  in  sputum,  60 
Adulteration,  meat,  precipitin  test 

for,  506 
Agar-agar,  glycerin,  preparation  of, 
460 
preparation  of,  460 
Air-bubbles  in  urine,  210 
Albumin  in  sputiun,  80 
Albuminometer,  Esbach's,  134 
Albuminuria,  128 
accidental,  128 
Boston's  test  in,  131 
cyclic,  129 
detection,  130 
Esbach's  estimation  of  albumin. 

134 

estimation  of  albimiin  m,  quan- 
titative, 133 

false,  128 

from  blood  changes,  129 

from  kidney  changes,  1 29 

heat  and  nitric  acid  test  in,  133 
test  in,  Purdy's,  133 

nitric  acid  test  in,  133 

orthostatic,  129 

physiologic,  128 

postural,  129 

Purdy's  centrifugal  method,  134 
heat  test  in,  133 
table  after  centrifugation,  135 

renal,  128 

666 


556 


INDEX 


Albuminuria,  Robert's  test  in,  131 

tests,  130-134 

trichloracetic  acid  test  in,  131 

Tsuchiya's   estimation   of   albu- 
min, 134 

Ulrich's  test  in,  133 
Alimentary  glycosuria,  138 
Alkaline  methylene-blue,  Loffler's, 

74 
phosphates  in  urine,  109 
urine,  unorganized  sediments  in, 
176,  184 
Alkalinity  of  urine,  92 
fixed,  92 
volatile,  92 
Alkapton  bodies  in  urine,  157 
Alkaptonuria,  157 
Alveolar  cells  in  sputum,  79 
Amboceptor,  495 

antisheep,  in  Wassermann  reac- 
tion, 512 
in  Wassermann  reaction,  titra- 
tion of,  516 
Amebae  in  feces,  348 
Amidobenzol  test  for  free  hydro- 
chloric acid,  326 
Ammonia  in  urine,  125 
Brown's  test,  127 
decreased,  126 
estimation,  quantitative,  126 
increased,  126 

Ronchese-Malfatti       formalin 
test,  127 
Ammoniated  silver  nitrate  solution, 

123,  124 
Ammoniomagnesium        phosphate 

crystals  in  urine,  185 
Ammonium  sulphate  test  for  globu- 
lin in  cerebrospinal  fluid,  428 
urate  crystals  in  lunne,  187 
Ammon's  bom,  451 
Amoeba  histolytica  in  sputum,  61 
Amorphous   phosphates   in    urine, 
109,  186 
in  mass,  197 
urates  in  urine,  90,  122,  178 
in  mass,  197 
Anaemia  infantum  pseudoleukaem- 

ica,  317 
Anemia,  308 
aplastic,  313 
blood-picture  in,  309 


Anemia,  blood-plaques  in,  259 
pernicious,  311 
color  index  in,  244 
degeneration  of  Grawitz  in,  274 
erythroblasts  in  blood  in,  276 
erythrocytes  in,  272 
leukopenia  in,  247 
lymphocytes  in,  280 
megaloblasts  in,  276 
myelocytes  in,  289 
polychromatophilia  in,  273 
posthemorrhagic,  310 
primary,  311 
secondary,  309 
splenic,  314 
von  Jaksch's,  317 
Anemias,  degeneration  of  Grawitz 
in,  274 
erythrocytes  in,  272 
normoblasts  in  blood  in,  276 
polychromatophilia  in,  273 
Angina,  Vincent's,  436 
spirochete  of,  374 
Anguillula,  403 
aceti,  403 
in  urine,  207 
Anilin-gentian  violet  stain,  74 
Animal  inoculation,  432 

method   for  tubercle  bacillus 

in  sputum,  69 
of  bacteria,  474 
parasites,  363.     See  also  Para- 
sites, animal. 
anemia  from,  309 
in  blood,  295 
in  feces,  352 
in  sputum,  61 
in  urine,  206 
Anisocytosis,  272 
Anopheles,  296,  299 
Antibodies,  219 
Antiformin    method    for    Bacillus 

tuberculosis  in  sputum,  68 
Antigen,  493 
in  Wassermann  reaction,  titra- 
tion of,  516 
syphilitic,  in  Wassermann  reac- 
tion, 511 
Antimeningococcus-sermn  test  for 
epidemic  cerebrospinal  meningi- 
tis, 429 
Antipyrin  in  urine,  166 


INDEX 


557 


Antisheep  amboceptor  in  Wasser- 

mann  reaction,  512 
Anuria,  88 
Aplastic  anemia,  313 
Apochromatic  objectives,  24 
Apothecaries'  measure,  553 

weight,  SS4 
Apparatus,  454,  545 
Arsenic  in  urine,  166 

Gutzeit's  test  for,  167 
Reinsch's  test  for,  166 
Arthropoda,  417 
Ascaris,  403 

canis,  405 

lumbricoides,  403 
ova  of,  404 

mystax,  403 
Aspirin  in  urine,  172 
Asthma,  bronchial,  eosinophilia  in, 
286 
sputum  in,  83 
Atrophic  gastritis,  gastric  contents 

in,  343 
Atropin  in  urine,  167 
Autoclave,  454 
Autogenous  vaccines,  479 
Avoirdupois  weight,  553 


Babcock    estimation    of    fat    in 

milk,  442 
Babesia,  382 

bigeminum,  382 

hominis,  382 
Bacillus,  acid-fast,  69 

Boas-Oppler,  in  gastric  contents, 

341 
colon,  477 
diphtheria,  477 

Neisser's  stain  for,  436 

Ponder's  stain  for,  436 
fusiform,  374 
Koch- Weeks,    in    conjunctivitis, 

438 
mucosus  capsulatus  in  sputimi, 

74 
of  Friedlander  in  sputum,  74 

of  influenza,  477 

in  cerebrospinal  fluid,  431 

in  sputum,  75 
pertussis  in  sputum,  76 
pyocyaneus  in  otitis,  440 


Bacillus  tuberculosis,  478 

in  cerebrospinal  fluid,  429 
m  feces,  359 
in  otitis,  440 
in  pus,  422 
in  sputum,  63,  83 
animal  inoculation  method, 

68 
antiformin  method,  68 
Gabbet's  method,  64 
Ldffler's  method,  68 
methods  for,  63-70 
Pappenheim's  method,  66 
Ziehl-Neelson  method,  65 
in  urine,  205 
detection,  205 
t3T)hosus,  477 
in  blood,  292 
technic,  293 
xerosis  in  eye,  439 
Bacteria,  animal  inoculation,  474 
characteristics  of,  475 
collection   of   material   for   cul- 
tural examination,  472 
cultural  methods  of  examining, 

472 
cultures  of,  study,  472 
Gram-negative,  468 
Gram-positive,  468 
in  blood,  292 

in  cerebrospinal  fluid,  429 
in  feces,  357 

stains  for,  358 
in  gastric  contents,  341 
in  milk,  441 
in  pus,  419 
in  sputum,  62 
in  urine,  91,  204 
incubation  of,  472 
inoculating  media  for,  472 
methods  of  studying,  471 
microscopic  examination,  471 
obtaining  of,  in  preparation  of 

vaccines,  481 
stains  for,  466 
Bacterial  casts  in  urine,  195 

vaccines,  479.    See  also  Vaccines. 
Bacterins,  479.    See  also  Vaccines. 
Bacteriologic  methods,  454 
Balantidiimi,  382 

coli,  382 
Basket-cells,  291 


558 


INDEX 


Basophilic  granular  degeneration, 
274 
leukocytes,  287 
structures  of  blood,  266 
Bass  and  Watkins'  modification  of 
macroscopic  Widal  reaction,  501 
B.  K.  tuberculin,  488 
Beef  extract  bouillon,  preparation 
of.  459 
infusion,  preparation  of,  459 
tapeworm,  392 
Bence-Jones  body,  136 

detection,  137 
Benedict's  estimation  of  glucose  in 
urine,  144 
test  for  glucose,  139 
Benzidin  test  for  blood,  307 

for  hemoglobin  in  urine,  157 
B.  F.  tuberculin,  488 
Bial's  orcinol  test  for  pentoses,  148 
Bile  acids  in  urine,  detection,  155 
Hay's  test  for,  155 
diminished   flow   of,   indican   in 

urine  from,  113 
in  feces,  353 
in  gastric  contents,  324 
in  urine,  154 

Gmelin's  test,  155 
Smith's  test,  155 
medium,  preparation  of,  463 
Bile -pigment   in   urine,   detection, 

154 
Bilharzia  haematobium,  387 
Bilharziasis,  387 

Biliousness,  indican  in  urine  in,  1 13 
Bilirubin  in  urine,  154 
Biologic  identification  of  unknown 
proteins,  503 
test  for  human  blood,  503 
for  meat  adulteration,  506 
Black  sputum,  52 
Bladder,  hemorrhage  from,  203 

Schistosomum  haematobium 
as  cause.  203 
Blood,  218 

acidophilic  structures  of,  266 
amount  of,  total,  219 
animal  parasites  in,  295 
Bacillus  t>'phosus  in,  292 

technic,  293 
bacteria  in,  292 
bacteriologic  study,  292 


Blood,  basophilic  structures  of,  266 
benzidin  test  for,  307 
biologic  test  for,  503 
carbol-thionin  stain  for,  271 
changes,  albuminuria  from,  129 

in  blood  diseases,  table,  318 
coagulation  of,  221 

Boggs'  method  of  estimating, 
223 

prevention,  222 

time,  222,  223 
color,  219 

index,  244 
crisis,  311 
diseases,  blood  change  in,  table, 

318 
Khrlich's  triple  stam  for,  266 
eosin  and  methylene-blue  for,  266 
eosinophilic  structures  of,  266 
er>'throcytes  in,  number,  233 
filarial  larvae  in,  304 
guaiac  test  for,  306 
Harlow's  stain  for,  269 
hemin  test  for,  307 
in  acute  myelogenous  leukemia, 

316 
in  anaemia  infantum  pseudoleu- 

kaemica,  317 
in  anemia,  309 

aplastic,  313 

pernicious,  311 

posthemorrhagic,  310 

primary,  311 

secondary,  309 

splenic,  314 
in  aplastic  anemia,  313 
in  chlorosis.  313 
in  chronic  lymphatic  leukemia, 

316 
in  feces.  350,  353 
in  gastric  contents,  324,  332,  340 

test  for,  333 
in  leukemia,  314 
in  lymphatic  leukemia,  316 
in  myelogenous  leukemia,  315 
in  pernicious  anemia,  311 
in  posthemorrhagic  anemia,  310 
in  splenic  anemia,  314 
in  sputum,  51 
in  urine,  90,  202,  203 
in  von  Jaksch's  anemia,  317 
Jenner's  stain  for,  271 


INDEX 


559 


Blood,  larvae  of  Trichinella  spiralis 
in,  305 

leiikocytes  in,  number,  247 

malarial  parasites  in,  296.     See 
also  Malarial  parasites. 

neutrophilic  structures  of,  266 

obtaining,  220 

oxyphilic  structures  of,  266 

Pappenheim's     pyronin-methyl- 
green  stain  for,  271 

parasites,  292 

pathology,  special,  308 

polychrome  methylene-blue-eosin 
stains  for,  267 

precipitin  test  for,  306,  503 

reaction,  219 

recognition  of,  tests  for,  306 

Spirochaeta  recurrentis  in,  295 

stains,  261 
for  films,  261 

Teichmann's  test  for,  307 

Trypanosoma  gambiense  in,  295 

typhoid  bacilli  in,  292 
technic,  293 

unstained,  malarial  parasites  in, 
301 

viscosity,  220 

volume  index,  245 

Larrabee's  method,  246 
method,  245 

watery,  219 

Wright's  stain  for,  267 
Blood-casts  in  urine,  195 
Blood-corpuscles  in  feces,  357 

red.     See  Erythrocytes. 

white,  218.    See  also  Leukocytes. 
Blood-dust  of  Miiller,  219 
Blood-films,  chemic  fixation,  264 

cigarette-paper  method,  263 

Ehrlich's  two  cover-glass  method, 
261 

fixing,  264 

heat  fixation,  265 

Kowarsky's  plate  for  fixing,  265 

making,  261 

malarial  parasites  in,  302 

spreading,  261 

stained,  study  of,  271 

staining,  261,  266 

stains  for,  261,  266 

two-slide  method,  262 
Blood-lancet,  Daland's,  220 


Blood-plaques,  218 

enumeration,  258 

in  anemiai  259 

in  infections,  259 

in  leukemia,  259 

in  purpura  haemorrhagica,  259 

Kemp-Calhoun-Harris      estima- 
tion, 259 

number,  258 

stained,  study  of,  291 

Wright  and  Kinnicutt's  estima- 
tion, 261 
Blood-platelets,  218 
Blood-serum,  219,  222 

Loffler's  preparation  of,  461 
Bluemel's  test  for  glycosuria,  140 
Boas'  reagent,  326 

test  for  free  hydrochloric  acid, 
326 

test-breakfast,  321 
Boas-Oppler  bacillus  in  gastric  con- 
tents, 341 
Bodo,  379 

urinarius,  379 
Boggs'  coagulation  instnunent,  223 

method  of  estimating  coagulation 
time  of  blood,  223 

modification  of  Esbach  method 
for  proteins  in  milk,  444 

throttle  control  for  blood-count- 
ing pipet,  25s       _ 
Boil,  Delhi,  Leishmania  tropica  of, 

378 
Borchardt's    test    for    levulose    in 

urine,  147 
Boric  acid  in  milk,  test  for,  445 
Boston's  method  for  keeping  semen 
for  examination,  449 

test  for  albumin,  131 
Bottles,  vaccine,  480 
Bouillon,  beef  extract,  preparation 

of,  459 
infusion,  preparation  of,  459 
Brick -dust  deposit  in  urine,  90 
Bromids  in  urine,  167 
Bronchi,   cylindric   cells   from,   in 

sputum,  79 
Bronchial  asthma,  eosinophilia  in, 
286 
sputum  in,  83 
casts  in  sputum,  53,  59 
Bronchiectasis,  sputum  in,  82 


56o 


INDEX 


Bronchitis,  sputum  in,  8i 
Brown's  test  for  ammonia  in  urine, 

127 
Bubbles  of  air  in  urine,  210 
Buerger's    method    for    pneumo- 
cocci  in  pus,  421 
for  pneumococcus  capsules,  72 
Burker's  hemacytometer,  242 
Butyric  acid   test,   Noguchi's,   for 

globuHn   in   cerebrospinal   fluid, 

428 


Cabot's  ring  bodies,  276 
Calcium  carbonate  in  urine,  187 

oxalate  in  urine,  179 
Calculus,  renal,  urine  in,  214 

vesical,  urine  in,  215 
Callison's  diluting  fluid,  484 
Calmette's     ophthalmo-tuberculin 

reaction,  490 
Cammidge's    pancreatic    reaction, 
163 
technic,  163 
Cane-sugar  in  urine,  148 
Capsules,  glass,  for  serum  work,  497 
of      pneumococcus,      Buerger's 
method  for,  72 
Carbolfuchsin,  65 
Carbol-gentian-violet,  70 
Carbol-thionin  stain  for  bacteria, 
467 
for  blood,  271 
Carcinoma,  gastric,  gastric  contents 
in,  343 
peptid-splitting  enzyme  in  gas- 
tric contents  in,  330 
Carriers,  dysentery,  370 
Casts,  bronchial,  in  sputum,  53,  59 
containing  organized   structures 

in  urine,  194 
fibrinous,  in  sputum,  60 
in  urine,  188 

negative-staining,  190 
Catarrh,  vernal,  eosinophilic  leuko- 
cytes in,  440 
Cells,  alveolar,  in  sputimi,  79 
basket-,  291 

compound  granular,  in  urine,  199 
cylindric,  in  sputum,  79 
eosinophilic,  in  sputum,  77 
epithelial,  in  feces,  357 


Cells,  ephthelial,  in  sputum,  78 

in  urine,  198,  199 
heart-failure,  in  sputum,  54,  79, 

82 
in  sputum,  76 
irregular,  in  urine,  199 
mast-,  287 
mesothelial,     predominance    of, 

426 
polyhedral,  in  urine,  198 
pus-,  in  gastric  contents,  340 
shadow,  in  urine,  202 
squamous,  in  sputum,  79 
vegetable,  in  feces,  356 
yeast-,  in  gastric  contents,  341 

in  urine,  208 
Centigrade  and  Fahrenheit  scales, 

554 
Central  illumination  of  microscope, 

20 
Centrifuge  for  serum  work,  496 
Purdy's,  106 

tube  for  milk  analysis,  443 
tubes,  Purdy's,  107 
water-motor,  106 
Cercomonas,  378 

hominis,  378 
Cerebrospinal  fluid,  ammonium  sul- 
phate test  for  globulin  in, 
428 
Bacillus  tuberculosis  in,  429 
bacteria  in,  429 
cytology  of,  431 
examination,  426 
chemic,  427 
macroscopic,  427 
microscopic,  429 
globulin  in,  427 

ammonium  sulphate  test  for, 

428 
Noguchi's  butyric  acid  test 
for,  428 
influenza  bacillus  in,  431 
Noguchi's  butyric  acid  test  for 

globulin  in,  428 
pneumococcus  in,  430 
sugar  in,  428 

Wassermann  reaction  with,  521 
meningitis,  epidemic,  430 

antimeningococcus      serum 
test  for,  429 
Cestoda,  384,  390 


INDEX 


561 


Cestodes,  390 

Charcot-Leyden  crystals  in  feces, 

359 
in  sputum,  58 
Chart,  Gibson's,  283,  284 
Chemic    examination    of    cerebro- 
spinal fluid,  427 
of  feces,  352 
of  gastric  contents,  325 
of  milk,  442 
of  sputum,  80 
of  mine,  100 
fixation  of  blood-films,  264 
Chemo taxis,  248 
Chloral  hydrate  in  urine,  167 
Chlorids  in  urine,  103 
detection,  103 

estimation,  Purdy's  centrifugal 
methods,  105 
table,  108 
quantitative,  104,  108 
Volhard's  method,  103 
in  nephritis,  103 
in  pneumonia,  103 
Chlorosis,  blood  in,  313 
color  index  in,  244 
leukopenia  in,  247 
lymphocytes  in,  280 
Chrysomyia  macellaria,  418 
Chyluria  from  filaria  infection,  184 
Cigarette-paper  method  for  blood- 
films,  263 
Cilia,  368,  382 
Cloudy  urine,  method  of  clearing, 

Coagulation,  221 

Bogg's  method  of  estimating,  223 

instnmient,  Boggs',  223 
Duke's,  222 

prevention  of,  222 

time,  222,  223 
Cobra- venom  test  for  syphilis,  528 
Coccidium,  381 

cuniculi,  381 
Cochin-China  diarrhea,  413 
Coin-like  disks  in  sputum,  84 
Colic,  mucous,  351 
Colon  bacillus,  477 
Color  index  of  blood,  244 
in  chlorosis,  244 
in  pernicious  anemia,  244 

of  blood,  219 

36 


Color  of  feces,  349 
of  sputvmi,  51 
of  urine,  89 
Complement,  495 
deviation  test  for  gonorrhea,  523 
for  malignant  disease,  524 
for  syphilis,  511 
for  tuberculosis,  526 
in  Wassermann  reaction,  513 
titration  of,  514 
Concretions  in  feces,  351 
Condenser  for  microscope,  23 
Congo-red   test   for   free   acids   in 

gastric  contents,  325 
Conjugate  sulphates  in  urine,  112 
Conjunctivitis,     acute     infectious, 
438. 
bacteria  of,  438 
diphtheric,  439 
pseudomembranous,  439 
Cook's  method  for  purin  bodies,  1 23 
Corpuscles,  blood-,  in  feces,  357 
red.    See  Erythrocytes. 
white,  218.     See  also  Leuko- 
cytes. 
pus-,  283,  419 
in  sputum,  76 
in  urine,  199 
predominance  of,  425 
Corrections  for  objectives,  25 
Cotton  fibers  in  urine,  197,  209 
fibrils  in  sputiun,  56 
sterilization  of,  458 
Craig's  table  for  differentiation  of 
Entamoeba  coli  and  Entamoeba 
histolytica,  371,  372 
Crisis,  blood,  311 
Croupous  pneumonia,  sputum  in, 

52,  83 
Cryoscopy  of  urine,  97 
Crystals,  Charcot-Leyden,  in  feces, 

359 
in  sputum,  58 

envelope,  in  urine,  179 

fatty-acid,  in  sputum,  56 

in  sputum,  59,  359 

lu^ic  acid,  in  urine,  177 
Culex,  299 
Cultural    methods    of    examining 

bacteria,  472 
Culture-media,  459 

preparation  of,  459 


562 


INDEX 


Culture-media,  reaction  of,  464 

sterilization  of,  457 

storage  of,  465 

tubing,  465 
Culture-tubes,  455 

preparation,  458 
Cultures  of  bacteria,  study,  472 
Curds  in  feces,  352 

of  milk  in  feces,  357 
Curschmann's  spirals  in  sputum,  57 
Curvature  of  microscopic  field,  27 
Cutaneous  test  for  syphilis,  491 
Cyclic  albuminuria,  129 
Cylindric  cells  in  sputum,  79 
Cylindroids  in  urine,  196 
Cylindruria,  188 
Cysticercus  cellulosae,  395 
Cystin  crystals  in  urine,  182 
Cystinuria,  182 
Cystitis,  urine  in,  215 
Cysts,  daughter-,  396 
Cytodiagnosis,  424 
Cytology  of  cerebrospinal  fluid,  431 


Daland's  blood-lancet,  220 
hematocrit,  246 

Dare's  estimation  of  hemoglobin, 
230 
hemoglobinometer,  230 

Dark-ground  illumination  of  micro- 
scope, 22 

Darling's  stain  for  Entamoeba  his- 
tolytica, 370 

Daughter-cysts,  396 

Decomposition  of  urine,  86 

Definitive  host  of  animal  parasites, 

364 
Degeneration  of  Grawitz,  274 
Delhi  boil,  Leishmania  tropica  of, 

378 
Demodex  folliculorum,  417 
Depth  of  focus,  30 
Desmoid   test,    Sahli's,  of   gastric 

digestion,  346 
Dextrose  in  urine,  137.     See  also 

Glycosuria. 
Diabetes  insipidus,  urine  in,  216 

melHtus,  urine  in,  216 
Diacetic  acid  in  urine,  153 

Gerhardt's  test  for,  153 
Lindemann's  test  for,  153 


Dialyzation  method  of  protective 

ferment  reaction,  532 
Dialyzing  membrane  for  protective 
ferment  reaction,  534 
testing,  534 
Diarrhea,  Cochin-China,  413 
Diazo-reaction,  159 
in  measles,  161 
in  tuberculosis,  160 
in  typhoid  fever,  160 
substitutes  for,  162 
technic,  161 
Diazo-substances  in  urine,  159 
Dibothriocephalus,  398 
latus,  391,  398,  402 
anemia  from,  309 
infection    with,    decrease    of 
hemoglobin  from,  225 
Dicalcium    phosphate    crystals   in 

urine,  185 
Dicroccelium,  386 
Diet,  Schmidt's,  for  examination  of 

feces,  360 
Digestion,  gastric,  Sahli's  test  for, 

346 
Digestive  leukocytosis,  250 
Dilatation  of  stomach,  gastric  con- 
tents in,  342 
Diluting  fluid,  Callison's,  484 
for  blood-plaque  count,  260 
fluids  for  blood  count,  240 
in  leukemia,  258 
Diphtheria  bacillus,  477 
Neisser's  stain  for,  436 
Ponder's  stain  for,  436 
Diphtheric  conjunctivitis,  439 
Diplobacillus  of  Morax  and  Axen- 

feld,  438 
Diplococcus,  Frankel's,  in  sputum, 

71 

intracellularis  meningitidis,  430, 
476 
Dipylidium,  398 

caninum,  398 
Dirt  on  slide  as  source  of  error,  210 
Dittrich's  plugs  in  sputum,  52 
Donne's  test  for  pus  in  urine,  90 
Doremus-Hinds'  ureometer,  116 
Dosage  of  tuberculin,  489 

of  vaccines,  486 
Drugs,  effect  of,  on  urine,  166 

leukocytosis  from,  252 


INDEX 


563 


Drugs,  resinous,  in  urine,  172 
Drunkard's  pneumonia,  sputum  in, 

51 

Dry  objective,  26 

Duke's  coagulation  instrument,  222 

Dungem's  test  for  malignant  dis- 
ease, 524 

Dunham's  peptone  solution,  prep- 
aration of,  463 

Dwarf  tapeworm,  397 

Dysentery  carriers,  370 
tropical,   Entamoeba  histolytica 
in,  368 


Ear,  440 

Earthy  phosphates  in  urine,  109 

Echinococcus  disease,  395 

diagnosis,  396 

eosinophilia  in,  286 
Edema,  pulmonary,  sputum  in,  81 
Edestin  test  for  gastric  contents, 

332. 
Eel,  vinegar,  403 

in  urine,  207 
Egyptian  hematuria,  203,  207,  387 
Ehrlich's  diazo-reaction,  159 

technic,  161 
side-rhain  theory  of  immunity, 

493 
triple  stain  for  blood,  266 
two-cover    method    for    blood- 
films,  260 
Einhom's  saccharimeter,  145 
Elastic  fibers  in  sputum,  54 
Electric  conductivity  of  urine,  97 
Elephantiasis,  406 
Embryos,  filarial,  in  blood,  304 
Empty  magnification,  32 
Endocarditis,  malignant,  vaccines 

in,  487 
Endomyces  albicans,  434 
Entamoeba,  368 
buccalis,  372 
coli,  370 

Entamoeba  histolytica  and,  dif- 
ferentiation, 371,  372 
histolytica,  368,  372 
Darling's  stain  for,  370 
EntamcEba  coli  and,  difiFeren- 
tiation,  371,  372 
tetragena,  372 


Enteritis,  membranous,  351 
Enteroliths  in  feces,  351 
Envelope  crystals  in  urine,  179 
Enzyme,  peptid-splitting,  in  gastric 
contents,  330 

solution,  preparation  of,  121 
Eosin     and     methylene-b'ue     for 

blood,  266 
Eosinophiles,  285 
Eosinophilia,  286 

in  bronchial  asthma,  286 

in  echinococcus  disease,  286 

in  filariasis,  286 

in  menstruation,  286 

in  myelogenous  leukemia,  286 

in  scarlet  fever,  286 

in  skin  diseases,  286,  287 

in  trichiniasis,  286 

in  uncinariasis,  286 

in  worm  infection,  286 
EosinophiUc  cells  in  sputimi,  77 

leukocytes,  285 

in  vernal  catarrh,  440 

structures  of  blood,  266 
Epidemic  cerebrospinal  meningitis, 

430 
antimeningococcus       serum 
test  for,  429 
Epithelial  casts  in  urine,  194 
cells  in  feces,  357 
in  sputum,  78 
in  urine,  198.  199 
Erythroblasts,  275 
Erythrocytes,  218,  233 
Biirker's  instrument  for  count- 
ing, 242 
counting  of,  234 
decrease  of,  233 
emmieration  of,  233 
in  anemias,  272 
in  chlorosis,  3r4 
in  gastric  contents,  340 
in  leukemia,  272 
in  pernicious  anemia,  272,  311 
in  sputum,  80 
in  urine.  202 
increase  of,  233 
number  of,  233 
pessary  forms,  272 
recognition  of,  tests  for,  306 
shape  of,  272 
size  of,  272 


5^4 


INDEX 


Erythrocytes,    stained,   study   of, 
272 
staining  properties,  variations  in, 

273 
Thoma-Metz      instrument      for 

counting,  243 
Thoma-Zeiss      instrument      for 

counting,  235 
variations  in  structure,  275 
Esbach's  albuminometer,  134 
estimation  of  proteins  in  milk, 

Boggs'  modification,  444 
method  for  albumin  in  urine.  134 
reagent  for  albuminuria,  134 
Estivo-autumnal  parasite,  296,  297, 

304 
Ethereal  sulphates  in  urine,  112 
Ewald's  salol  test  for  gastric  motor 
power,  346 

test-breakfast,  321 
Exercises,  practical,  in  microscopy, 

42-47 
Exhausting     diseases,     secondary 

anemia  from,  309 
Exophthalmic  goiter,  lymphocytes 

in,  280 
Exudates,  423 

decomposition    of,     indican    in 
urine  from,  113 
Eye,  438 
Eye-pieces,  microscopic,  24 


Fahrenheit  and  Centigrade  scales, 

554 
False  albuminuria,  128 
Fasciola,  385 

hepatica,  385 
Fasciolopis,  386 

buski,  386 
Fat  in  feces,  357 

in  milk,  estimation,  442,  443 

in  urine,  182 
Fat-droplets  in  sputum,  80 

in  urine,  184,  193,  199,  209 
Fat-globules  in  urine,  182 
Fatty  casts  in  urine,  193 
Fatty-acid  crystals  in  sputiun,  56 

needles  in  sputum,  59 
Favus,  441 
Feces,  348 

acholic,  350 


Feces,  amebae  in,  348 
animal  parasites  in,  352 
Bacillus  tuberculosis  in,  359 
bacteria  in,  357 

stains  for,  358 
bile  in,  353 
blood  in,  350,  353 
blood-corpuscles  in,  357 
Charcot-Leyden  crystals  in,  359 
chemic  examination,  352 
color  of,  349 
concretions  in,  351 
consistence,  349 
curds  in,  352 

of  milk,  357 
enterohths  in,  351 
epithelial,  in  feces,  357 
examination,  chemic,  352 

macroscopic,  349 

microscopic,  355 

specimen  for,  348 
fat  in,  357 

food  particles  in,  356 
form,  349 

frequency  of  passage,  349 
functional  tests,  360 
motility,  362 
Sahli's  glutoid,  362 
Schmidt's  diet,  360 
nuclei,  361 
gall-stones  in,  351 
hydrobilirubin  in,  354 
macroscopic  examination,  349 
maggots  in,  417 
microscopic  examination,  355 
mucus  in,  351 
occult  hemorrhage  in,  detection, 

353 
odor,  350 
ova  in, 360 
parasites  in,  360 
quantity,  349 
pus  in, 357 

starch-granules  in,  356 
tapeworms  in,  352 
trypsin  in,  354 

Gross'  test  for,  354 
vegetable  cells  in,  356 

fibers  in,  356 

hairs  in,  356 
Fehling's  estimation  of  glucose  in 
urine,  143 


INDEX 


565 


Fehling's  test  for  glucose,  139 
Ferment  diagnosticum,  331 
reactions,  protective,  530 
Abderhalden's,  530 
dialyzation  method,  532 
polariscopic  method,  540 
preparation   of  organ  pep- 
tone, 540 
proving   the   peptone   solu- 
tion, 542 
Fermentation,  acid,  of  urine,  91 
method  of  estimating  glucose  in 
urine,  145 
Fibers,  elastic,  in  sputum,  54 
of  cotton  in  urine,  197,  209 
of  linen  in  urine,  209 
of  muscle  in  sputum,  357 

in  mine,  210 
of  silk  in  urine,  209 
of  wool  in  urine,  197,  209 
vegetable,  in  feces,  356 
Fibrils,  cotton,  in  sputum,  56 
Fibrinous  casts  in  sputum,  60 

in  urine,  103 
Filaria,  406 
bancrofti,  406 
diuma,  408 

infection,  chyluria  from,  184 
loa,  408 
medinensis,  408 
perstans,  408 
philippinensis,  408 
sanguinis  hominis,  407 
Filarial  lar\'ae  in  blood,  304 
Filariasis,  eosinophilia  in,  286 
Filariform  larvae,  414 
Fischer's  test-meal,  322 
Fish  tapeworm,  398 
Fixation,   chemic,   for  blood-films, 
264 
heat,  of  blood-films,  265 
of  blood-films,  264 
Flagella,  LofHer's  stain  for,  470 
Flagellata,  367,  372 
Flasks,  455 
Flat  worms,  384 

Flaws  in  slide  as  source  of  error,  210 
Fleischl-Miescher     estimation     of 
hemoglobin,  228 
hemoglobinometer,  228 
Fleischl's  estimation  of  hemoglobin, 
226 


Fleischl's  hemoglobinometer,  226 
Floaters  in  urine,  206 
Florence's    reaction    for   detection 
of  semen,  450 
reagent,  451 
Fluke's,  384 
liver,  385 
lung,  387 
Focus,  depth  of,  30 
Folin's  method  of  estimating  urea 
in  lu-ine,  121 
of  quantitative  estimation  of 
acidity  of  urine,  92 
Food  particles  in  feces,  356 

in  gastric  contents,  324,  339 
Formaldehyd    in    urine,    Rimini- 

Bumam  test  for,  168 
Formalin  in  milk,  test  for,  445 
Formalin-gentian- violet  stain,  74 
Frankel's  diplococcus  in  sputum,  71 
Free    acids    in    gastric    contents, 
Congo-red  test  for,  325 
tests  for,  325 
hydrochloric  acid,  319.    See  also 
Hydrochloric  acid,  free. 
Freezing-point  of  urine,  determina- 
tion, 97 
Friedlander's  bacillus  in  sputum,  74 
Friedman    and   Hamburger's    test 

for  gastric  contents,  332 
Frommer's  test  for  acetone,  152 
Frothingham's  method  of  demon- 
strating Negri  bodies,  451,  452 
modification    of    Van    Gieson's 
stain  for  Negri  bodies,  452 
Fniit  sugar  in  urine,  146 
Functional         tests,         digestion, 
Schmidt's  test  diet,  360 
for  feces,  360 
motility,  362 
Sahli's  glutoid,  362 
Schmidt's  diet,  360 
nuclei,  361 
for  urine,  97 
intestines,  motility,  362 
kidney,  cryoscopy,  97 
electric  conductivity,  97 
methylene-blue  test,  98 
phenolsulphonephthalein 

test,  98 
phloridzin  test,  98 
liver,  Strauss  levulose  test,  147 


566 


INDEX 


Functional    tests,   liver,   urobilin, 

159 
jjancreas,  Sahli's  glutoid  test, 
362 

Schmidt's  nuclei  test,  361 

trypsin,  354 
stomach,     absorptive    power, 

344 
Sahli's  desmoid  test,  346 
motor  power,  345 
Fungus,  mold,  in  urine,  209 

ray-,  in  sputum,  60 
Funnel,    separatory,    for    Strauss' 

lactic  acid  test,  328 
Fusiform  bacillus,  374 


Gabbet's     method     for     Bacillus 
tuberculosis  in  sputum,  64 
stain,  65 

GafTky's  table  for  recording  num- 
ber of  tubercle  bacilli  in  sputum, 
67 

Gall-stones  in  feces,  351 

Gametes  in  malaria,  298 

Gangrene  of  lung,  sputum  in,  82 

Gastric  carcinoma,  gastric  contents 

in,  343 
peptid-splitting  enzyme  in  gas- 
tric contents  in,  330 
contents,  acid  deficit,  337 
bacteria  in, ^41 
bile  in,  324 
blood  in,  324,  332,  340 

test  for,  333 
Boas-Oppler  bacillus  in,  341 
chemic  examination,  325 
edestin  test  for,  332 
erythrocytes  in,  340 
examination,  319 

chemic,  325 

microscopic,  339 

physical,  323 

routine,  320 
food  particles  in,  324,  339 
free  acids  in,  Congo-red  test 
for,  325 
tests  for,  325 
Friedman    and    Hamburger's 

test  for,  332 
hydrochloric  acid  in,  319.    See 

also  Hydrochloric  ctcid,  free. 


Gastric  contents  in  achylia  gas- 
trica,  343 
in  atrophic  gastritis,  343 
in  carcinoma,  343 
in  chronic  gastritis,  343 
in  dilatation,  342 
in  disease,  342 
in  gastric  ulcer,  344 
in  gastrosuccorrhea,  343 
in  neuroses,  342 
lactic  acid  in,  326.    See  also 

Lactic  acid. 
Leptothrix  buccalis  in,  341 
microscopic  examination,  339 
mucus  in,  324 
obtaining,  320 
organic  acids  in,  326 

quantitative  tests,  337 
pepsin    in,     328.      See    also 

Pepsin  in  gastric  contents. 
pepsinogen  in,  328 

test  for,  329 
peptid-splitting  enzj^ne  in,  330 
physical  examination,  323 
pus-cells  in,  340 
reaction,  324 
rennin  in,  330 
test  for,  330 
renninogen  in,  330 

test  for,  330 
sarcinae  in,  340 
tests,  qualitative,  325 

quantitative,  333 
tissue  bits  in,  324 
total  acidity,  333 
tests,  333 
Topfer's  test,  333 
withdrawal,  322 
yeast-cells  in,  341 
digestion,  Sahli's  test  for,  346 
neuroses,  stomach  contents  in, 

342 
ulcer,  gastric  contents  in,  344 
Gastritis,  atrophic,  gastric  contents 
in,  343 
chronic,  gastric  contents  in,  343 
Gastrosuccorrhea,  gastric  contents 

in,  343 
Gauze,  sterilization  of,  458 
Gelatin,  preparation  of,  461 
Gerhardt's  test  for  diacetic  acid, 
153 


INDEX 


567 


Gibson's  chart,  283,  284 
Giemsa's  stain  for  syphilis,  447 
Glass  capsules  for  serum  work,  497 
Glassware,  sterilization  of,  457 
Globular  sputum,  84 
Globules,  myelin,  in  sputimi,  80 
Globulin  in  cerebrospinal  fluid,  427 
ammonium  sulphate  test  for, 

428 
Noguchi's  butyric  acid  test 
tor,  428 
Glossina  morsitans,  377 

palpalis,  2q6 
Glucose  in  urine,   137.     See  also 

Glycosuria. 
Glutoid  test,  Sahli's,  for  digestive 

functions,  362 
Glycerin  agar-agar,  preparation  of, 

460 
Glycosuria,  137 
alimentary,  138 

Benedict's  quantitative  estima- 
tion, 144 
test  in,  139 
Bluemel's  test  in,  140 
detection  of  dextrose,  138 
estimation  of  glucose,  141 
Fehling's    quantitative    estima- 
tion, 143 
test  in,  139 
fermentation  method  of  estimat- 
ing, 14s 
Haine's  test  in,  138 
Kowarsky's  test  in,  140 
persistent,  138 
phenylhydrazin  test  in,  140 
Purdy's  estimation  of  glucose,  142 
Robert's  differential  method  of 

estimating,  146 
transitory,  137 
GmeUn's  test  for  bile,  155 
Goiter,  exophthalmic,  lymphocytes 
in,  280 
lymphocjrtes  in,  280 
Gonococcxis,  476 
in  ophthalmia,  439 
in  pus,  422 
in  urine,  206 
Gonorrhea,  complement  deviation 
test  for,  523 
Schwartz  and  McNeil's  test  for, 
523 


Gk)norrheal  ophthalmia,  439 

threads  in  urine,  196,  206 
Gram-negative  bacteria,  468 
Gram-positive  bacteria,  468 
Gram's  iodin  solution,  74 

method  for  pus,  419 

stain  for  bacteria,  467 
Granular  casts  in  urine,  193 

degeneration,  basophilic,  274 
Granules,  lycopodium,  in  urine,  210 

Much,  in  sputimi,  69 
Frontes'  method,  70 
methods  for,  70 

Schiiffner's,  275 

starch,  in  luine,  209 
Gravel  in  urine,  177 
Grawitz's  degeneration,  274 
Gray  sputimi,  52 
Gross'   test  for   trypsin   in  feces, 

354 
Ground  itch,  411 
Guaiac  test  for  blood,  306 

for  hemoglobin,  157 
Guinea-worm,  408 
Gunning's  test  for  acetone,  151 
Gutzeit's  test  for  arsenic  in  urine, 

167 


Haines'  solution,  139 

test  for  glucose,  138 
Hairs  in  urine,  197 

vegetable,  in  feces,  356 
Hall's  method  for  purin  bodies,  123 

purinometer,  124 
Hammer's  test  for  tuberculosis,  526 
Hammerschlag's      estimation      of 
hemoglobin,  230 

test  for  pepsin,  338 
Harlow's  blood  stain,  269 
Hart's  test  for  oxybutyric  acid,  154 
Haser's  method  for  total  solids  in 

urine,  96 
Hayem's  fluid  for  blood  count,  240 

hematoblasts,  292 
Hay's  test  for  bile-acids,  155 
Heart-failure  cells  in  sputum,  54, 

79,  82 
Heat  and  nitric  acid  test  for  albu- 
min, 133 

fixation  of  blood-films,  265 

test  for  albvunin,  Pvuyd's,  133 


568 


INDEX 


Hemacytometer,  Burker's,  242 

Thoma-Metz,  243 

Thoma-Zeiss,  235 

cleaning  instrument,  241 
sources  of  error,  240 
technic,  237 
Hematemesis,  hemoptysis  and,  dif- 
ferentiation, 324 
Hematoblasts  of  Hayem,  292 
Hematocrit,  Daland's,  246 
Hematuria,  203 

Egyptian,  203,  207,  387 

hemoglobinuria  and,  difTerentia- 
tion,  156 

idiopathic,  203 
Hcmin  test  for  blood,  307 
Hemoglobin,  218,  224 

Dare's  estimation,  230 

decrease,  225 

from   infection   with    Diboth- 
riocephalus  latus,  225 

estimation,  225 

Fleisch-Miescher  estimation,  228 

Hammerschlag's  estimation,  230 

in  pernicious  anemia,  311 

increase,  225 

medium,  preparation  of,  462 

Sahli's  estimation,  228 

Tallqvist's  estimation,  232 

Von  Fleischl's  estimation,  226 
Hemoglobinometer,  Dare's,  23c 

Fleischl-Miescher,  228 

Sahli,  228 

Tallqvist's,  232 

von  Fleischl's,  225 
Hemoglobinuria,  156 

benzidin  test  in,  157 

detection,  156 

guaiac  test  in,  157 

hematuria    and,    differentiation, 
156 

Teichmann's  test  in,  156 
Hemolytic  system,  511 
Hemoptysis,  hematemesis  and,  dif- 
ferentiation, 324 
Hemorrhage,  anemia  from,  308 

from  bladder,  203 

Schistosomum  haematobium 
as  cause,  203 

occult,  in  feces,  detection,  353 
Herpetomonas,  378 
Hexamethylenamin  in  urine,  168 


Hiss'  method  for  pneumococci  in 
pus,  421 
serum-media,  preparation  of,  463 
Holt's  milk- testing  apparatus,  442 
Hookworm,  American,  409,  410 
anemia  from,  309 
infection,  409 

diagnosis,  411 
New  World,  409 
Old  World,  409 
Horismascope,  131,  132 
Host,   defmitive,   of  animal   para- 
sites, 364 
intermediate,  of  animal  parasites, 
364 
Hot-air  sterilizer,  454 
Huntoon's  stain  for  spores,  469 
Hyaline  body,  296 

casts  in  urine,  188,  190  . 
Hydatid  disease,  395 

diagnosis,  396 
Hydrobilirubin  in  feces,  354 
Hydrochloric  acid,  combined,  319, 

335 
Topfer's  test  for,  336 
free,  319 
absence,  335 

amidobenzol  test  for,  326 
Boas'  test  for,  326 
decrease,  335 
increase,  335 
tests  for,  325 

quantitative,  334 
Topfer's  test  for,  336 
Hydrogen  sulphid  generator,  170 
Hydrophobia,  451.      See  also  Ra- 
bies. 
Hymenolepis,  397 
diminuta,  398 
nana, 397 
Hyperchlorhydria,  335 
Hyperchromemia,  225 
Hyperemia,  renal,  active,  urine  in, 
211 
passive,  urine  in,  212 
urine  in,  211 
Hyphae  of  molds  in  urine,  198 
Hypobromite  method  of  estimating 

urea  in  urine,  117 
Hypochlorhydria,  335 
Hy^jodermic  injection  of  tuberculin 
in  diagnosis,  490 


INDEX 


569 


Idiopathic  hematuria,  203 

polycythemia,  225 
Illumination,  dark-ground,  of  mi- 
croscope, 22 
for  microscope,  18 

\vith    water-bottle    condenser, 

Image,  microscopic,  27 
Immersion  objective,  26 
Immune  bodies,  494 

of     second     order,     reactions 

based  upon,  497 
of  third  order,  reactions  based 
upon,  510 
Immunity,  493 
acquired,  493 
amboceptor,  495 
complement,  495 
Ehrlich's  side-chain  theory,  493 
receptors,  494 
Incidental  parasites,  383 
Incubation  of  bacteria,  472 
Incubator,  455 
Index,  color,  of  blood,  244 
opsonic,  508.     See  also  Opsonic 

index. 
volume,  of  blood,  245 

Larrabee's  method,  246 
method,  245 
India-ink  method  for  sjT)hilis,  448 
Indican  in  urine,  112 
detection,  113 

from    decomposition    of    exu- 
dates, 113 
from  diminished  flow  of  bile, 

"3 
in  biliousness,  113 
in  diseases  of  small  intestine, 
112 
of  stomach,  113 
Obermayer's  test  for,  113 
Indophenol  oxydase  test  for  myelo- 
blasts, 290 
Infections,  blood-plaques  in,  259 
leukocytosis  in,  251 
secondary  anemia  from,  309 
vaccines  in,  487 
Inflammation,  leukocytosis  in,  251 
pseudomembranous,    of    mouth, 

435 
Influenza  bacillus,  477 

in  cerebrospinal  fluid,  431 


Influenza  bacillus  in  sputimi,  75 

Infusion,  beef,  preparation  of,  459 
bouillon,  preparation  of,  459 

Infusoria,  368,  382 

Inoculating  media  for  bacteria,  472 

Inoculation,  animal,  432 

for  tubercle  bacillus  in  sputum, 

69 
of  bacteria,  474 

Intermediate  host  of  animal  para- 
sites, 364 

Intestine,  small,  diseases  of,  indi- 
can in  urine  in,  112 

Intoxication,  acid,  cause,  149 

lodin  in  urine,  168 

reaction  of  leukocytes,  285 
solution.  Gram's,  74 

lodophilia,  285 

Irregular  malaria,  297 

Itch,  ground,  411 
mite,  417 


Jaksch's  anemia,  317 
Jenner's  blood  stain,  271 


Kala-azar,  Leishmania  donovani 
of,  378 

Kelling's  test  for  lactic  acid,  328 

Kemp-Calhoun-Harris     estimation 
of  blood-plaques,  259 

Kidney  changes,  albuminuria  from, 
129 

Koch-Weeks   bacillus   in   conjunc- 
tivitis, 438 

Kowarsky's   plate   for   fixation   of 
blood-films,  264 
test  for  glucose,  140 


Lactic  acid  in  gastric  contents,  326 
Kelling's  test  for,  328 
Simon's  test  for,  328 
Strauss'  test  for,  328 
Uffelmann's  test  for,  327 

Lactose  in  milk  estimation,  444 
in  urine,  148 

Lamblia,  380 
intestinalis,  380 

Lamp,  Matthews'  microscope,  20 

Lancet,  blood-,  220 


57° 


INDEX 


Lange's  test  for  acetone,  152 
Larrabee's    estimation   of    volume 

index  of  blood,  246 
Larvae,  filarial,  in  blood,  304 

filariform,  414 

in  sputvmi,  62 

of  Trichinella  spiralis  in  blood, 

305 
rhabditiform,  413 
Lead  in  urine,  168 

Lederer's  test,  169 
Lead-poisoning,  chronic,  degenera- 
tion of  Grawitz  in,  274 
Lederer's  test  for  lead  in  urine,  169 
LeiTmann-Beam  estimation  of  fat 

in  milk,  441 
Leg.il's  test  for  acetone,  Lange's 

modification,  152 
Leishman-Donovan  bodies,  378 
Leishmania,  378 
donovani,  378 
infantum,  378 
tropica,  378 
Leishman's  method  of  determining 

opsonic  index,  508 
Leptothrix  buccalis,  433 
in  gastric  contents,  341 
in  sputum,  56 
Leucin  in  urine,  180 
Leukemia,  247,  253,  314 
blood-plaques  in,  259 
degeneration  of  Grawitz  in,  274 
erythrocytes  in,  272 
leukocyte  count  in,  253 
lymphatic.  316 
acute,  317 
chronic,  316 
leukocytes  in,  280 
myelogenous,  315 
acute,  316 
eosinophilia  in,  286 
mast-cells  in,  288 
myeloblasts  in,  290 
myelocytes  in,  289 
Neubauer  ruling  for  blood  count 

in,  253 
Todd's  estimation  of  leukocytes 

in,  256 
Tiirck's  ruling  for  blood  count  in, 

253 
Zappert  ruling  for  blood  count 
in,  253 


Leukocytes,  218 
abnormal  varieties,  288 
atypic  forms,  290 
basophilic,  287 
border-line  forms.  290 
classification  of,  278 
counting,  differential,  277 

in  leukemia,  253 
decrease  in,  247 
degenerated  forms.  291 
enumeration  of,  246 
eosinophilic,  285 

in  vernal  catarrh,  440 
in  pernicious  anemia,  312 
increase  in,  247 

absolute,  278 

relative,  278 
iodin  reaction  of,  285 
irritation  forms,  291 
mononuclear,  large,  280 
normal,  279 
number  of,  246 

polymorphonuclear  neutrophilic, 
281 

predominance  of,  425 
polynuclear,  282 
stained,  study  of,  277 
transitional,  281 
vacuolated,  291 
Leukocytosis,  247,  248 
digestive,  250 
lymphocytic,  252 

in  hereditary  syphilis,  253 

in  pertussis,  253 
permanent,  247 
polymorphonuclear,  249 

from  drugs,  252 

from  infections,  251 

from  inflammations.  251 

in  malignant  disease,  252 

pathologic,  250 

physiologic,  249 

posthemorrhagic,  252 

toxic,  252 
transient,  247 
Leukopenia,  247 
in  chlorosis,  247 
in  pernicious  anemia,  247 
lymphocytes  in,  280 
Levulose  in  urine,  146 

Borchardt's  test,  146 

detection.  147 


INDEX 


571 


Levulose  in  urine,  quantitative  es- 
timation, 146 

Lieben's  test  for  acetone,  Gunning's 
modification,  152 

Lindemann's  test  for  diacetic  acid, 

Linen  fibers  in  urine,  209 
Linguatula  serrata,  418 
Litmus  milk,  preparation  of,  462 
Liver  fluke,  385 

rot,  38s 
Loffler's   alkaline    methylene-blue, 

74 
blood-serum,  preparation  of,  461 
method  for  Bacillus  tuberculosis 

in  sputum,  68 
methylene-blue  for  gonococci  in 
pus,  422 
for  pus,  419 
stain  for  flagella,  470 
Louse,  417 
Luetin,  491 

skin  test  for  syphilis,  491 
Lung  fluke,  387 

gangrene  of,  sputum  in,  82 
Lycopodium  granules  in  urine,  210 
Lymphatic  leukemia,  316 
acute,  317 
chronic,  316 
lymphocytes  in,  280 
Lymphocytes,  279 

predominance,  425 
Lymphocytic  leukocytosis,  252 
in  hereditary  syphilis,  253 
in  pertussis,  253 
Lymphocytosis,  252 


Macrocytes,  272 
Macroscopic  examination  of  cere- 
brospinal fluid,  427 
of  sputum,  50 
Maggots  in  feces,  417 
Magnesia  mixture,  124 
Magnification,  empty,  32 
microscopic,  31 

methods  of  increasing,  32 
Malaria,  irregular,  297 

large  mononuclear  leukocjrtes  in, 

281   _    _ 
transmission  of,  by  mosquitoes, 
298 


Malarial  parasites,  296 

cycles,  296 

detection,  300 

estivo-autumnal,  296,  297,  304 

gametes  in  blood  in,  298 

in  blood-films  in,  302 

in  unstained  blood,  301 

life  histories,  296 

merozoites  of,  296 

mosquitoes  as  hosts,  298 

quartan,  296,  297,  304 

Ruge's  stain  for,  302 

segmentation,  296 

sexual  cycle,  296,  298 

spores  of,  296 

stains  for,  362 

tertian,  296,  297,  304 

Wright's  stain  for,  302 
strippling,  274 
Malignant     disease,     complement 
deviation  test  for,  524 

leukocytosis  in,  252 

von  Dungem's  test  for,  524 
endocarditis,  vaccines  in,  487 
tumors,  anemia  from,  309 
Maltose  in  urine,  148 
Mast-cells,  287 
Mastigophora,  367,  372 
Matthews'  microscope  lamp,  19,  20 
Measles,  diazo-reaction  in,  161 
Measures,  553 
Meat  adulteration,  precipitin  test 

for,  506 
Media,    culture-,    459.      See    also 

Culture-inedia. 
Megaloblasts,  275 
Megalocytes,  272 
Melanin  in  urine,  158 

tests  for,  158 
Melanuria,  158 

Membrane,  dialyzing,  for  protect- 
ive ferment  reaction, 

534 
testmg,  534 
Membranous  enteritis,  351 
Meningitis,  cerebrospinal,  epidemic, 

430 
antimeningococcus       serum 

test  for,  429 
Menstruation,  eosinophilia  in,  286 
Mercury  in  urine,  170 
Merozoites  of  malarial  parasites,  296 


572 


INDEX 


Mesothelial    cells,    predominance, 

426 
Metal,  sterilization  of,  457 
Methylene-bluc  test  for  urine,  98 

Russo's,  162 
Metric  system,  553 
Mett's  test  for  {pepsin,  338 

tubes,  339 
Microblasts,  275 
Micrococcus  catarrhalis  in  sputum, 

75-  76 
Microtytes,  272 

Micrometer    eye-piece    for    micro- 
scope, 37,  38 

stage,  38 
Micron,  40 
Microscope,  17 

care  of,  36 

choice  of,  42 

cleaning,  36 

condenser  for,  23 

curvature  of  field,  27 

eye-pieces  for,  24 

illumination  for,  18 
dark-ground,  22 

lamp,  Matthews',  20 

magnification  by,  31 

methods  of  increasing,  32 

micrometer  eye-piece  for,  37,  38 

objectives  for,  24 
corrections,  25 

useof,  17,  33 
Microscopic   examination   of   cere- 
brospinal fluid,  429 
of  feces,  355 
of  sputum,  53 

image,  27 

objects,  measurement  of,  37 
Micturition,  frequency  of,  87 
Milk,  441 

analysis  of,  441 
tube  for,  443 

bacteria  in,  441 

boric  acid  in,  test  for,  445 

chemical  examination,  442 

curds  of,  in  sputum,  357 

fat  in,  estimation,  442,  443 

formalin  in,  test  for,  445 

lactose  in,  estimation,  444 

litmus,  preparation  of,  462 

preservatives  in,  detection,  444 

proteins  in,  estimation,  442,  444 


Milk  reaction,  441 

Milk-sugar  in  urine,  148 

Milk-testing  apparatus,  Holt's,  442 

Milky  urine,  90 

Mineral  sulphates, in  urine,  112 

Miracidium,  389 

Mite,  itch,  417 

Mold  fungi  in  urine,  209 

Molds,  hyphae  of,  in  urine,  198 
in  sputum,  61 

Moller's  stain  for  spores,  469 

Mononuclear  leukocytes,  large,  280 

Morax  and  Axenfeld's  diplobacil- 
lus,  438 

Momer's  test  for  tyrosin,  182 

Moro's  tuberculin  reaction  in  diag- 
nosis of  tuberculosis,  490 

Morphin  in  urine,  171 

Morphology-,  staining  for,  466 

Mosquito  in  transmission  of  mala- 
ria, 298 

Motor  power  of  stomach,  345 

Mouth,  diseases  of,  433 
organisms  of,  433 

Much  granules  in  sputiun,  6g 
Frontes'  method,  70 
methods  for,  70 

Mucin  in  urine,  136 

Mucous  colic,  351 
threads  in  urine,  195 

Mucus  in  feces,  351 
in  gastric  contents,  324 

Miiller's  blood-dust,  219 

Muscle,  fibers  of,  in  urine,  210 

Muscle-fibers  in  sputum,  357 

Myelin  globules  in  sputum,  80 

Myeloblasts,  290 

indophenol  oxydase  test  for,  290 

Myelocytes,  288 

Alyelogenous  leukemia,  314 
acute,  316 
eosinophilia  in,  286 
mast-cells  in,  288 
myeloblasts  in,  290 
myelocytes  in,  289 


Negator  americanus,  409,  410 
Needles,    fatty-acid,    in    sputum, 

59 
Negative  staining  of  tuinary  casts, 
190 


INDEX 


573 


Negri  bodies,  451 

Frothingham's  method  of  de- 
monstrating, 451,  452 
Van  Gieson's  stain  for  Froth- 
ingham's modification,  452 

Neisser's  stain  for  diphtheria  bacil- 
lus, 436 

Nemathelminthes,  383,  402 

Nematoda,  402 

Nematodes,  402 

Nephritis,  chlorids  in  urine  in,  103 
urine  in,  21 1-2 13 

Neubauer  ruling  for  count  in  leu- 
kemia, 253 

Neuroses,  gastric,  stomach  con- 
tents in,  342 

Neutrophilic  leukocytes,  polymor- 
phonuclear, 281 
structures  of  blood,  266 

Nitrogen  equilibrium,  114 
in  urine,  114,  115 
partition,  114 

Noguchi's  butyric  acid  test  for 
globulin  in  cerebrospinal  fluid, 
428 

Normoblasts,  275 

Nose,  cylindric  cells  from,  in  spu- 
tum, 79 

Nubecula  of  urine,  90 

Nuclei  test,  Schmidt's,  for  pan- 
creatic insufficiency,  361 

Numeric  aperture,  28 

Nutrition,  poor,  secondary  anemia 
from,  309 

Obermayer's  reagent,  114 

test  for  indican  in  urine,  113 
Objectives,  achromatic,  24 

apochromatic,  24 

depth  of  focus,  30 

dry,  26 

immersion,  26 

method  of  attaching,  35 

microscopic,  24 

numeric  apertures,  28 

oil-immersion,  26 

resolving  power  of,  28 

working  distance  of,  27 
Oblique  illumination  of  microscope, 

20 
Occult  hemorrhage  in  feces,  detec- 
tion, 353 


Oil-immersion  objective,  26 
Oligochromemia,  225 
Oligocythemia,  233 
Oliguria,  88 
Oncospheres,  391 
Ophthalmia,  gonorrheal,  439 
Ophthalmotuberculin  reaction,  Cal- 

mette's,  490 
Opisthorchis,  386 

felineus,  386 

sinensis,  386 
Oppenheim   and    Sachs'   stain  for 

syphilis,  449 
Opsonic  index,  508 

Leishman's  method  of  deter- 
mining, 508 
Wright's  method  of  determin- 
.ing,  507 
Opsonins,  507 
Organ  peptone,  preparation  of,  for 

protective  ferment  reaction,  540 
Organic  acids  in  gastric  contents, 
326 
quantitative  tests,  337 
Oriental  sore,  Leishmania  tropica 

of,  378 
Orthostatic  albuminuria,  129 
Otitis,  440 

bacteria  of,  440 
Ova  in  feces,  360 
Oxybutyric  acid  in  urine,  154 

Hart's  test  for,  154 
Oxyphilic  structures  of  blood,  266 
Oxyuris,  405 

vermicularis,  405 

Pancreatic  insufficiency, 

Schmidt's  nuclei  test  for,  361 
reaction,  163 

in  pancreatitis,  163 
technic,  163 
Pancreatitis,    pancreatic    reaction 

in,  163 
Pappenheim's  method  for  Bacillus 
tuberculosis  in  sputum,  66 
pyronin-methyl-green    for    bac- 
teria, 467 
for  gonococci  in  pus,  422 
for  pus,  419 
stain  for  blood,  271 
Paragonimus,  387 
kellicotti,  387 


574 


INDEX 


Paragonimus  ringeri,  387 
westermannii,  387 
in  sputum,  6i 
Paramoecium  coli,  382 
Parasites,  animal,  363 
anemia  from,  309 
classification,  365 
defmitive  host,  364 
in  blood,  295 
in  feces,  352 
in  sputum,  61 
in  urine,  206 
intermediate  host,  364 
nomenclature,  365 
blood,  292 
in  feces,  360 
incidental,  383 

malarial,  296.    See  also  Malarial 
parasites. 
Parasitic  diseases  of  skin,  441 
Paratyjjhoid  fever,  Widal  reaction 

in,  497 
Pathologic  polymorphonuclear  leu- 
kocytosis, 250 
Pavement  epithelial  cells  in  urine, 

199 
Pediculus  capitis,  417 
pubis,  417 
vestimenti,  417 
Pentoses  in  urine,  148 

Bial's  orcinol  test,  148 
Pepper's  method  of  concentration 

of  ova  of  hookworm,  413 
Pepsin  in  gastric  contents,  328 

Hammerschlag's  test,  338 
Mett's  test,  338 
quantitative  test,  337 
Schiitz's  law,  338 
test  for,  329,  337 
Pepsinogen  in  gastric  contents,  328 

test  for,  329 
Peptid-splitting  enzyme  in,  330 
Peptone,  organ,  preparation  of,  for 
protective    ferment   reactions. 
540 
solution,  Dunham's  preparation 
of,  463 
proving,  for  protective  ferment 
reaction,  542 
Pericardial  fluid,  examination,  423 
Peritoneal  fluid,  examination,  423 
Permanganate  test,  Weisz's,  162 


Pernicious  anemia,  311 

Cabot's  ring  bodies  in,  277 

color  index  in,  244 

degeneration  of  Grawitz  in,  274 

erythroblasts  in  blood  in,  276 

erythrocytes  in,  272 

leukopenia  in,  247 

lymphocytes  in,  280 

megaloblasts  in,  276 

myelocytes  in.  289 

polychromatophilia  in,  273 
Persistent  glycosuria,  138 
Pertussis,  lymphocytes  in,  280 

lymphocytic  leukocytosis  in,  253 
Pessary  forms  of  erythrocytes,  272 
Phagocytic  index,  508 
Phagocytosis,  507 
Pharyngomycosis  leptothrica,  434 
Pharynx,    tuberculous    ulceration, 

437       .     . 
Phenacetin  in  urine,  166 
Phenol  in  urine,  171 
Phenolphthalein  in  urine,  172 
Phenolsulphonephthalein    test    for 

urine,  98 
Phenylhydrazin    test    for   glucose, 

140 
Phloridzin  test  for  urine,  98 
Phosphates  in  urine,  109,  184 
acid  calcium,  185 
alkaline,  109 
amorphous,  90,  109,  186 

in  mass,  197 
decreased,  no 
earthy,  109 

estimation,  quantitative,  no 
increased,  109 
Purdy's  table  for,  after  cen- 

trifugation,  no 
stellar,  186 
triple,  109,  185 
Phosphaturia.  109 
Photomicrography,  40 
Physiologic  albuminuria,  128 
polymorphonuclear  leukocytosis, 
249 
Pigments  in  urine,  87,  89 
Pink-eye,  438 
Pin-worm,  405 
Pipets,  456 

for  serum  work,  497 
Piroplasma  hominis,  382 


INDEX 


575 


Pirquet's  reaction  in  tuberailosis, 

490 
Plasmodiiun,  381,  382 

falciparum,  296 

malariae,  296.    See  also  Malarial 
parasites. 

tenue,  296 

vivax,  296 
Platinum  wires,  455 
Platyhelminthes,  383,  384 
Pleural  fluid,  examination,  423 
Plugs,  Dittrich's,  in  sputimi,  52 
Pneumococcus,  476 

capsules,  Buerger's  method  for, 

.72 

in  cerebrospinal  fluid,  430 

in  eye  affections,  438 

in  pus,  421 

in  sputmn,  71 

Smith's  method,  73 
Pneiunonia,  chlorids  in  urine  in, 
103 

croupous,  sputiun  in,  52,  83 

drunkards',  sputum  in,  51 
Poikilocytes,  272 
Poikilocytosis,  272 
Pointer,  27 

Poisoning,  chronic,  secondary  ane- 
mia from,  309 

lead-,   chronic,   degeneration  of 
Grawitz  in,  274 
Polariscopic  method  of  protective 

ferment  reaction,  540 
Polychromatophilia,  273 
Polychrome    methylene-blue-eosin 

stains  for  blood,  267 
Polycythemia,  233 

idiopathic,  225 
Polyhedral  cells  in  urine,  198 
Polymorphonuclear  leukocytes,  pre- 
dominance, 425 

leukocytosis,  249.    See  also  Leu- 
kocytosis, polymorphonuclear. 

neutrophilic  leukoc)rtes,  281 
Polynuclear  leukocytes,  282 
Polyuria,  88 

Ponder's  stain  for  diphtheria  bacil- 
lus, 436 
Pork  tapeworm,  394 
Posthemorrhagic  anemia,  310 

leukocytosis,  252 
Postural  albuminuria,  129 


Potassium    indoxyl     sulphate    in 
urine,    112.     See   also   Indican 
in  urine. 
Potato  medium,  preparation  of,  462 
Power  of  resistance,  283 
Precipitin  test  for  blood,  306,  503 

for  meat  adulteration,  506 
Preformed  sulphates  in  urine,  112 
Preservatives   in   milk,   detection, 

444 
Proglottides,  391 

Progressive  pernicious  anemia,  311 
Protective  ferment  reactions,  530 
Abderhalden's,  530 
dialyzation  method,  532 
polariscopic  method,  540 
preparation  of  organ  pep- 
tone, 540 
proving   the  peptone  solu- 
tion, 542 
Proteins  in  milk,  estimation,  442, 

444 
in  virine,  128 

imknown,  biologic  identification, 
502 
precipitin  test  for,  503 
•Proteoses  in  urine,  136 
Protozoa,  366,  367 
Prune-juice  sputum,  51 
Pseudocasts  in  urine,  197 
Pseudomembranous  conjimctivitis, 

439 
inflammations  of  mouth,  435 
Pulmonary  edema,  sputimi  in,  82 
tuberculosis,  lymphocytes  in,  280 
sputum  in,  83 
Purdy's  centrifugal  estimation  of 
albiunin,  134 
of  chlorids,  105 
of  phosphates,  109 
of  sulphates,  iii 
centrifuge  tubes,  107 
electric  centrifuge,  106 
estimation  of  glucose  in  urine, 

142 
heat  test  for  albiunin,  133 
solution  for  glucose  test,  143 
table  for  estimation  of  albumin, 

13s 
of  chlorids,  108 
of  phosphates,  1 10 
of  sulphates,  iii 


576 


INDEX 


Purin  bodies  in  urine,  122 
Cook's  method,  123 
estimation,  quantitative,  123 
Purinometcr,  Hall's,  124 
Purpura      ha;morrhagica,      blood- 
plaques  in,  259 
Pus,  Bacillus  tuberculosis  in,  422 

bacteria  in,  419 

examination  of,  419 

gonococci  in,  422 

Gram's  method  for,  419 
Jn  feces,  357 

in  gastric  contents,  340 

in  urine,  90,  200 
Donne's  test,  90 

Lofller's  methylene-blue  for,  419 

Pappenheim's     pyronin-methyl- 
green  for,  419 

pneumococci  in,  421 

staphylococci  in,  420 

streptococci  in,  420 
Pus-casts  in  urine,  195 
Pus-cells  in  gastric  contents,  340 
Pus-corpuscles,  283,  419 

in  sputum,  76 

in  urine,  199 

predominance,  425 
Pyelitis,  urine  in,  215 
Pyuria,  200 


Quartan  parasite,  296,  297,  304 
Quinin  in  urine,  172 


Rabies,  diagnosis  of,  451 

Frothingham's    method    of    de- 
monstrating Negri  bodies  in, 

451,  452 

Ray-fungus  in  sputum,  60 

Reaction,  diazo-,  159 
pancreatic,  163 

Wassermann,     511.       See     also 
Wasscrmann  reaction. 

Reagents,  547 

Receptors  of  first  order,  494 
of  second  order,  494 
of  third  order,  494 

Red    blood-corpuscles.      See    Ery- 
throcytes. 

Reinsch's  test  for  arsenic  in  urine, 
166 


Relapsing  fever,  spirochetes  of,  373 
Renal  albuminuria,  128 

calculus,  urine  in,  214 

hyperemia,  urine  in,  211 

tuberculosis,  urine  in,  212 
Rennin  in  gastric  contents,  330 

test  for,  330 
Renninogen  in  gastric  contents,  330 

test  for,  330 
Resinous  drugs  in  urine,  172 
Resistance,  power  of,  283 
Resolving  power  of  objective,  28 
Rhabditiform  larva;,  413 
Rhizopoda,  367,  368 
Rice's  solutions,  118 
Riegel's  test-meal,  321 
Rimini-Burnam    test    for    formal- 

dehyd  in  urine,  168 
Ring  bodies  of  Cabot,  276 
Ringworm,  441 

Robert's  differential  density  meth- 
od of  estimating  glucose  in 
urine,  146 

test  for  albumin,  131 
Ronchese-Malfatti     formalin     test 

for  ammonia  in  urine,  127 
Round  worms,  402 
Rowntree  and  Geraghty's  phenol- 

sulphonephthalein  test  for  urine, 

Ruge's  stain  for  malarial  parasites, 

302 
Ruhemann's  method  for  uric  acid, 
125 

reagent,  125 

uricometer,  125 
Russo's  methylene-blue  test,  162 
Rusty  sputum,  51,  52 


Saccharimeter,  Einhom's,  145 
Sahli's  desmoid  test  of  gastric  di- 
gestion, 346 
estimation  of  hemoglobin,  228 
glutoid  test  for  digestive  func- 
tions, 362 
hemogloblnometer,  228 
Salicylates  in  urine,  172 
Salol  in  urine,  172 
test,  Ewald's,  for  gastric  motor 
power,  346 
Sand,  red,  in  urine,  177 


INDEX 


577 


Sarcinae  in  gastric  contents,  340 

Sarcodina,  367,  .368 

Sarcoptes  scabiei,  417 

Saxe's  urinopyknometer,  94,  95 

Scarlet  fever,  eosinophilia  in,  286 

Schistosomum,  385,  387 

haematobium,  387 
as  cause  of  hemorrhage  from 

bladder,  203 
in  urine,  206 

japonicum,  390 

mansoni,  389 
Schlesinger's   test   for   urobilin   in 

urine,  159 
Schmidt's  diet  for  examination  of 
feces.  360 

nuclei  test  for  pancreatic  insuf- 
ficiency, 361 
Schuffner's  granules,  275 
Schiitz's  law  in  quantitative  test 

for  pepsin,  338 
Schwartz  and    McNeil's    reaction 

for  gonorrhea,  523 
Scolex,  391,  392 
Scratches   on   slide   as   source   of 

error,  210 
Screw  worm,  418 
Secondary  anemia,  309 
Sediment,  urinary,  173.     See  also 

Urinary  sediment. 
Segmentation  of  malarial  parasites, 

296 
Semen,  examination  of,  449 

on  clothes,  detection,  449 
Florence's  reaction  for,  450 
Separatory  funnel  for  Strauss'  lac- 
tic acid  test,  328 
Serodiagnostic  methods,  493 

apparatus,  495 
Serosomucin  in  exudates,  423 
Serum,  blood-,  219,  222 

Loffler's  preparation  of,  461 

media.  Hiss'  preparation  of,  463 
Serum-albumin  in  urine,  1 28 
Serum- globulin  in  urine,  128 
Shadow  cells  in  urine,  202 
Silk  fibers  in  urine,  209 
Silver    impregnation    method    for 
syphilis,  448 

nitrate     solution,     ammoniated, 
123,  124 
Simon's  test  for  lactic  acid,  328 

37 


Skin  diseases,  eosinophilia  in,  286, 
287 
parasitic  diseases  of,  441 
test  for  syphilis,  491 
Sleeping  sickness,  296 

Trypanosoma    gambiense    of, 
376 
Smith's  method  for  pneumococci 
in  pus,  421 
in  sputum,  73 
test  for  bile,  155 
Solids,  total,  in  urine,  94 
Haser's  method,  96 
Specific  gravity  of  urine,  93 
Spermatozoa,  absence  of,  449 

in  urine,  204 
Spirals,  Curschmann's,  in  sputum, 

57 
Spirochaeta,  373 

buccalis,  374 

carteri,  373 

dentium,  374,  375 

duttoni,  373 

kochi,  373 

novyi,  373 

pallida,  445 

recurrentis,  373 
in  blood,  295 

refringens,  375,  446 

vincenti,  373 
Splenic  anemia,  314 
Splenomegaly,      infantile,      Leish- 

mania  infantum  of,  378 
Spores,  Huntoon's  stain  for  469 

MoUer's  stain  for,  469 

of  malarial  parasites,  296 
Sporozoa,  368,  381 
Sputum,  48 

Actinomyces  bovis  in,  60 

albumin  in,  80 

alveolar  cells  in,  79 

Amoeba  histolytica  in,  61 

animal  parasites  in,  61 

Bacillus  mucosus  capsulatus  in, 

of  influenza  in,  75 

pertussis  in,  76    . 

tuberculosis  in,  63,  83 
bacteria  in,  62 
black,  52 
blood  in,  51 
bronchial  casts  in,  53,  59 


:>/< 


INDEX 


Sputum,  cells  in,  76 

Charcot-Lcyden  crystals  in,  58 
chemic  c.\aminatii)n,  80 
coin-like  disks  in,  84 
collecting   sample   for   examina- 
tion, 48 
color,  51 
consistence,  52 
cotton  fibrils  in,  56 
crudum,  52 
crystals  in,  59,  359 
Curschmann's  spirals  in,  57 
cylindric  cells  in,  79 
Dittrich's  plugs  in,  52 
elastic  fibers  in,  54 
eosinophilic  cells  in,  77 
epithelial  cells  in,  78 
cry-throcytes  in,  80 
examination,  48 

chemic,  80 

collecting  sample  for,  48 

macroscopic,  50 

microscopic,  53 

physical,  51 

routine,  49 

fat-droi)lets  in,  80 
fatty-acid  cr>'stals  in,  56 

needles  in,  59 
fibrinous  casts  in,  60 
I'^ninkel's  dijjlococcus  in,  71 
Friedlander's  bacillus  in,  74 
globular,  84 
Kray,  52 

heart-failure  cells  in,  54,  79,  82 
in  bronchial  asthma,  83 
in  bronchiectasis,  82 
in  bronchitis,  81 
in  croupous  i)neumonia,  52,  83 
in  disease,  8i 

in  drunkards'  jineumonia,  51 
in  gangrene  of  lung,  82 
in  i)ulmonary  edema,  82 

tuberculosis,  83 
larvae  in,  62 

Leptothrix  buccalis  in,  56 
Micrococcus    catarrhalis   in,   75, 

76 
molds  in,  61 
Much  granules  in,  69 
Frontes'  method,  70 
methods  for,  70 
muscle-fibers  in,  357 


Sputum,  myelin  globules  in,  80 

Paragonimus  westermannii  in,  61 

I)neumococcus  in,  71 
Smith's  method,  73 

prune-juice,  51 

pus-corpuscles  in,  76 

quantity,  51 

ray-fungus  in,  60 

receptacle  for,  49 

red  blood-corjiuscles  in,  80 

rusty,  51,  52 

scjuamous  cells  in,  79 

stained,  62 

staphylococcus  in,  70 

streptococcus  in,  70 

Streptothrix  actinomyces  in,  61 

unstained,  53 

yeasts  in,  61 
Squamous  cells  in  sputum,  79 

epithelial  cells  in  urine,  199 
Squibb's  urinometer,  93 
Stage  micrometer,  38 
Stained  blood,  261 

sjuitum,  62 
Staining  for  morpholog>%  466 

methods,  465 
Stains,  547,  550 

anilin-gentian-violet,  74 

carbol-thionin,  for  bacteria,  467 
for  blood,  271 

Darling's,  for  Entamoeba  histo- 
lytica, 370 

Ehrlich's  triple,  for  blood,  266 

eosin    and    methylene-blue,    for 
blood,  266 

for  bacteria,  466 
in  feces,  358 

for  blood,  261 

for  blood-films,  261,  266 

for  diphtheria  bacillus,  436 

for  malarial  parasites,  302 

for  pus,  419 

for  syphilis,  447 

formalin-gentian-violet,  74 

Gabbet's,  65 

Giemsa's,  for  sj'philis,  447 

Gram's,  for  bacteria,  467 
for  pus,  419 
iodin  solution,  74 

Harlow's,  for  blood,  269 

Huntoon's,  for  spores,  469 

India-ink,  for  sj'philis,  448 


INDEX 


579 


Stains,  Jenner's,  for  blood,  271 
Loffler's  alkaline  methylene-blue, 

74 
for    Bacillus    tuberculosis    in 

sputum,  68 
for  flagella,  470 
methylene-blue,  for  gonococci 
in  pus,  422 
for  pus,  419 
MoUer's,  for  spores,  469 
negative,  for  urinary  casts,  190 
Neisser's,  for  diphtheria  bacillus, 

436 
Oppenheim  and  Sachs',  for  syph- 
ilis, 449 
Pappenheim's,  for  tubercle  bacil- 
lus in  sputum,  66 
pyronin-methyl-green,  for  bac- 
teria, 467 
for  blood,  271 
for  gonococci  in  pus,  422 
for  pus,  419 
polychrome  methylene-blue- 

eosin,  for  blood,  267 
Ponder's,  for  diphtheria  bacillus, 

436 
Ruge's,    for   malarial   parasites, 

.302 

silver,  for  syphilis,  448 
Van  Gieson's,  for  Negri  bodies, 
Frothingham's      modification, 
452 
Wright's,  for  blood,  267 
for  malarial  parasites,  302 
for  syphilis,  448 
Ziehl-Neelson,  65 
Staphylococci  in  pus,  420 

in  sputum,  70 
Staphylococcus   in   eye   affections, 
438 
pyogenes  albus,  475 
aureus,  475 
citreus,  475 
Starch-granules  in  feces,  356 

in  vunne,  209 
Starch-paper,  345 
Steam  sterilizer,  454 
Stellar  phosphates  in  urine,  186 
Sterility,  449 
Sterilization,  457 

in  preparation  of  vaccines,  482 
of  cotton,  458 


Sterilization  of  culture-media,  457 

of  gauze,  458 

of  glassware,  457 

of  metal,  457 
Sterilizers,  454 

dry,  454 

hot-air,  454 

steam,  454 
Stock  vaccines,  479 
Stomach,  319 

absorptive  power  of,  344 

contents  of,  319.    See  also  C(Z5/nc 
contents. 

digestion,  319 

dilatation  of,  gastric  contents  in, 

342 

diseases  of,  indican  in  urine  in,  1 13 

motor  power  of,  345 

position  of,  determination,  346 

size  of,  determination,  346 

worm,  405 
Stools,  348.    See  also  Feces. 
Storage  of  culture-media ,  465 
Strauss'  test  for  lactic  acid,  328 
Streptococci  in  pus,  420 

in  sputum,  70 
Streptococcus    in    eye    affections, 

438,  439 
pyogenes,  475 
Streptothrix   actinomyces   in   spu- 
tum, 61 
Strippling,  malarial,  274 
Strongyloides,  413 

intestinalis,  364,  413 
Substrate   for   protective    ferment 

reactions,  532 
Sugar  in  cerebrospinal  fluid,  428 

media,  preparation  of,  461 
Sugars  in  urine,  137 
Sulphates  in  urine,  1 1 1 
conjugate,  112 

estimation,  Purdy's  centrifugal 
method,  iii 
table  after  centrifugation, 
III 
quantitative,  in 
ethereal,  112 
mineral,  112 
preformed,  112 
Syphilis,  cobra- venom  test  for,  528 
complement  deviation   test  for, 


58o 


INDEX 


Syphilis,  cutaneous  test  for,  491 
dark -ground  illumination  in,  449 
examination  of  material,  445 
Giemsa's  stain  for,  447 
hereditary-,    l>-mphocytic    leuko- 
cytosis in,  253 
India-ink  method  for,  448 
luetin  skin  test  for,  490 
micro-organisms  of,  445,  446 
Oppenhcim  and  Sachs'  stain  for, 

449 
silver  impregnation  method  for, 

448 
Spirochaita  pallida  in,  445 

refringens  in,  446 
Treponema  pallidum  in,  445,  446 

stains  for,  447 
Wassermann   reaction   for,    511. 
See  also  Wusscnuann  reaction. 
Weil's  test  for,  528 
Wright's  stain  for,  448 
Syphilitic  antigen  in  Wassermann 

reaction,  511 
Syringe  for  serum  work,  497 


T.^NiA,  392 

echinococcus,  365,  392,  395 
in  urine,  206 

elliptica,  398 

saginata,  365,  391.  392 

solium.^  391,  392,  394 
Tallqvist's    estimation    of    hemo- 
globin, 232 

hemoglobinometer,  232 
Tannin  in  urine,  172 
Tapeworm,  384,  390 

beef,  392 

dwarf,  397 

fish,  398 

in  feces,  352 

pork,  394 
Teichmann's  test  for  blood,  307 

for  hemoglobinuria,  156 
Telosporidia,  368,  381 
Temperature,  554 
Tertian  parasite,  296,  297,  304 
Test-breakfast,  Boas',  321 

Ewald's,  321 
Test-meals,  321 

Boas',  321 

Ewald's,  321 


Test-meals,  Fischer's,  322 

Riegel's,  321 
Test-tube  rack  for  serum  work,  496 
Texas  fever,  Babesia  bigeminum  of, 

382 
rhoma-Metz  hemacytometer,  243 
Thoma-Zeiss  hemacytometer,  235 
cleaning  instrument,  241 
sources  of  error,  240 
technic,  237 
Thorn-apple  cr>'stals  in  urine,  187 
Thread- worm,  405 
Thrombin,  222 
Thrush,  434 

Tick-fever,  Babesia  hominis  of,  382 
Tinea  versicolor,  441 
Tissue  biis  in  gastric  contents,  324 
T.  O.- tuberculin,  488 
Todd's  estimation  of  leukocytes  in 

leukemia,  256 
Toisson's  fluid  for  blood  count,  240 
Topfer's  test  for  combined  hydro- 
chloric acid,  336 
for  free  hydrochloric  acid,  336 
Toxic  absorption,  degree  of,  283 

leukocytosis,  252 
Toxocara  canis,  405 
T.  R.  tubercuHn,  488 
Trachea,    cylindric   cells   from,   in 

sputum,  79 
Trachoma,  440 
Transitional  leukocytes,  281 
Transitorj'  glycosuria,  137 
Transudates,  423 
Trematoda,  384 
Trematodes,  384 
Treponema,  375 

pallidum,  375,  445,  446 
Giemsa's  stain  for,  447 
India-ink  method,  448 
Oppcnheim   and   Sachs'    stain 

for,  449 
silver    impregnation    method, 

448 
Wright's  stain  for,  448 
pertenue,  376 
Trichinella,  414 
spiralis,  364.  414 

lar\-ffi  of,  in  blood,  305  • 
Trichiniasis,  diagnosis,  415 
eosinophilia  in,  286 
parasite  of,  414 


INDEX 


581 


Trichloracetic  acid  test  for  albu- 
minuria, 131 
Trichocephalus,  416 

trichiurus,  416 
Trichomonas.  379 

intestinalis,  380 

pulmonalis,  380 

vaginalis,  380 
in  urine,  207 
Triple  phosphates  in  urine,  109,  185 
Tropical  dysentery,  Entamoeba  his- 
tolytica in,  368 
Trypanosoma,  376 

brucei,  377 

cruzi,  377 

equiperdum,  377 

evansi,  377 

gambiense,  376 
in  blood,  295 

lewisi,  377 

rhodesiensi,  377 
Trypanosomes,  376 
Trypsin  in  feces,  354 

Gross'  test  for,  354 
Tsuchiya's  method  for  albumin  in 

urine,  133 
Tube-casts  in  urine,  188 
Tubercle    bacillus.      See    Bacillus 

tuberculosis. 
Tuberculin,  488 

B.  E.,  488 

B.  P.,  488 

dosage  of,  489 

in  diagnosis,  490 

Calmette's       ophthalmo-reac- 

tion,  489 
hypodermic  injection,  490 
Moro's  reaction,  490 
von  Pirquet's  reaction,  490 

in  tuberculosis,  488 

reaction  of,  489 

T.  O.,  488 

T.  R.,  488 
Tuberculosis,    complement    devia- 
tion test  for,  526 

diazo-reaction  in,  i6c 

Hammer's  test  for,  527 

of  mouth,  436 

of  pharynx,  437 

pulmonarj--,  lymphocytes  in,  280 
sputum  in.  83 

renal,  urine  in,  212 


Tuberculosis,  tuberculin  in,  488 

vesical,  urine  in,  215 
Tubing  culture-media,  465 
Tumors,  malignant,  anemia  from, 

3.09 
vesical,  urine  in,  215 
Turck's  ruling  for  blood  coimt  in 

leukemia,  253 
Two-slide  method  for  blood-films, 

262 
Typhoid  bacillus,  477 
in  blood,  292 
technic,  293 
fever,  diazo-reaction  in,  160 
lymphocytes  in,  280 
vaccines  in,  488 
Widal  reaction  in,  497 
Tyrosin  in  urine,  180 

Momer's  test  for,  182 


Uffelmann's  -test  for  lactic  acid, 

327 

Uhlenhuth's  test  for  unknown  pro- 
teins, 503 

Ulcer,  gastric,  stomach  contents  in, 

344 
Ulceration  of  mouth,  437 

of  pharynx,  437 
Ulrich's  test  for  albumin.  133 
Uncinaria  duodenalis,  409 
Uncinariasis,  eosinophilia  in,  286 
Unstained  blood,  malarial  parasites 
in,  301 
sputum,  53 
Urate     crystals,     ammonium,     in 

urine,  187 
Urates,   amorphous,   in  urine,  90, 
122,  178 
in  mass,  197 
Urea  in  urine,  114 
decreased,  115 

estimation,    Folin's    method, 
121 
hypobromite  method,  117 
quantitative,  117 
urease  method,  118 
increased,  115 
retention,  115 
Urease  method  of  estimating  urea 

in  urine,  118 
Ureometer,  Doremus-Hinds',  116 


582 


INDEX 


Urethral  shreds  in  urine,  196 
Uric  acid  crystals  in  urine,  177 
in  urine,  122 

Cook's  mclhod,  123 
endogenous,   122 
estimation,  quantitative,  124 
exogenous,  122 
Hall's  method,  12,^ 
Ruhcmann's  method,  125 
Uricomctcr,  Ruhcmann's,  125 
Urinary  sediment,  examination,  173 
organized,  187 
unorganized,  175 

in  acid  urine,  176,  177 
in  alkaline  urine,  176,  184 
Urine,  85 

acetanilid  in,  166 

acetone     in,      149.       See     also 

Acetoniiriii. 
acid,  unorganized  sediments  in, 

176,  177 
acidity  of,  91 

quantitative  estimation,  92 
Folin's  method,  92 
air-bubbles  in,  210 
albumin     in,     128.       See     also 

Albuminuria. 
alkaline,  unorganized  sediments 

in,  176,  184 
alkalinity  of,  92 
fixed,  92 
volatile,  92 
alkapton  bodies  in,  157 
ammonia  in,  125.    See  also  Am- 
monia in  urine. 
ammoniomagnesium     phosphate 

crystals  in,  185 
ammonium  urate  crystals  in,  187 
amorphous  phosphates  in,  90 
urates  in,  90,  122,  178 
in  mass,  197 
Anguillula  aceti  in,  206 
animal  parasites  in,  206 
antipyrin  in,  166 
arsenic  in,  166 

Gutzeit's  test  for,  167 
Reinsch's  test  for,  166 
aspirin  in,  172 
atropin  in,  167 
bacteria  in,  91,  204 
bacterial  casts  in,  195 
Bence- Jones  body  in,  136 


Urine,   Bence-Jones  body  in,  de- 
tection, 137 

bile  in,   154.     See  also  Bile  in 
urine. 

bile-acids  in,  detection,  155 
Hay's  test  for,  155 

bile-pigment  in,  detection,  154 

bilirubin  in,  154 

blood  in,  90,  202,  203 

blood-casts  in,  195 

blood-corpuscles  in,  202 

brick-deposit  in,  90 

bromids  in,  167 

bubbles  of  air  in,  210 

calcium  carbonate  in,  187 
oxalate  in,  179 

cane-sugar  in,  148 

casts  in,  188 

containing     organized     struc- 
tures, 194 
negative  staining,  190 

chemic  examination,  100 

chloral  hydrate  in,  167 

chlorids     in,     103.       See     also 
Chlorids  in  urine. 

cloudy,  method  of  clearing,  87 

collection  of  specimen,  86 

color,  89 

composition,  86,  loi 

constituents,  abnormal,  127 
inorganic,  loi 
organic,  loi 

cryoscopy  of,  97 

cylindroids  in,  196 

cystin  crystals  in,  182 

decomposition  of,  86 

decreased,  88 

dextrose  in,  137.    See  also  Glyco- 
suria. 

diacetic  acid  in,  153.     See  also 
Diacelic  acid  in  urine. 

diazo-substances  in,  159 

dicalcium  phosphate  crystals  in, 

185 
effect  of  drugs  on,  166 
electric  conductivity,  97 
envelope  crystals  in,  179 
epithelial  casts  in,  194 

cells  in,  198,  199 
erythrocytes  in,  202 
examination,  chemic,  icxj 

microscopic,  173 


LNDEX 


583 


Urine,  examination,  physical,  88 
extraneous  structures  in,  208 
fat  in,  182 

fat-droplets  in,  184,  193,  199,  209 
fat-globules  in,  182 
fatty  casts  in,  193 
fibers  in,  of  cotton,  197,  209 

of  linen,  209 

of  muscle,  210 

of  silk,  209 

of  wool,  197,  209 
fibrinous  casts  in,  193 
fixed  alkalinity  of,  92 
floaters  in,  206 
formaldehyd  in,  Rimini-Bumam 

test  for,  168 
freezing-point  of,  determination, 

97 
fruit  sugar  in,  146 
functional  tests  for,  97 
glucose  in,  137.    See  also  Glyco- 
suria. 
gonococci  in,  206 
gonorrheal  threads  in,  196,  206 
granular  casts  in,  193 
gravel  in,  177 
hairs  in,  197 
hemoglobin   in,    156.      See   also 

Hemoglobinuria. 
hexamethylenamin  in,  168 
hyaline  casts  in,  188,  190 
hyphae  of  molds  in,  198 
identification  of,  87 
in  cystitis,  215 
in  diabetes  insipidus,  216 

mellitus,  216 
in  disease,  210 
in  nephritis,  211-213 
in  pyelitis,  215 
in  renal  calculus,  214 

hyperemia,  211 

tuberculosis,  212 
in  vesical  calculus,  215 

tuberculosis,  215 

tumors,  215 
increased,  88 
indican  in,  112.   See  also  Ituiican 

in  urine. 
inorganic  constituents,  101 
iodin  in,  168 

irregular  epithelial  cells  in,  199 
lactose  in,  148 


Urine,  lead  in,  168 

Lederer's  test.  169 
leucin  in,  180 
levulose  in,  146 

Borchardt's  test,  146 

detection,  147 

quantitative  estimation,  147 
lycopodium  granules  in,  210 
maltose  in,  148 
melanin  in,  158 

tests  for,  158 
mercury  in,  170 
methylene-blue  test  for,  08 
microscopic  examination,  173 
milk-sugar  in,  148 
milky,  90 
mold  fungi  in,  200 
morphin  in,  171 
mucin  in.  136 
mucous  threads  in,  195 
nitrogen  in,  114,  115 
normal  constituents,  ico 
nubecula  of,  90 
odor,  91 

organic  constituents,  loi 
oxybutyric  acid  in,  154 
Hart's  test  for,  154 
pavement  epithelial  cells  in,  199 
pentoses  in,  148" 

Bial's  orcinol  test,  148 
phenacetin  in,  166 
phenol  in,  171 
phenolphthalein  in,  172 
phenolsulphonephthalein  test  for, 

98 
phloridzin  test  for,  98 
phosphates  in,  109, 184.    See  also 

Piiosphaies  in  urine. 
physical  examination,  88 
pigments  in,  87,  89 
polyhedral  cells  in,  198 
potassium  indoxyl  sulphate  in, 

112.      See    also    Indican    in 

urine. 
preservation  of,  86 
proteins  in,  128 
proteoses  in,  136 
pseudocasts  in,  197 
purin  bodies  in,  122 

Cook's  method,  123 
estimation,  quantitative,  123 
pus  in,  91,  200 


584 


INDEX 


Urine,  pus  in,  Donne's  test,  90 
pus-casts  in,  195 
pus-coqniscles  in,  199 
quantity  passed,  88 
quinin  in,  172 
reaction,  91 
red  blood-corpuscles  in,  202 

sand  in,  177 
resinous  drugs  in,  172 
retention  with  overflow,  87 
salicylates  in,  172 
salol  in,  172 
Schistosomum   haematobium   in, 

206 
serum-albumin  in,  128 
serum-globulin  in,  128 
shadow  cells  in,  202 
solids  in,  total,  94 

Haser's  method,  96 
specific  gravit\-,  93 
si)ecimen  of,  collection,  86 
spermatozoa  in,  204 
squamous  epithelial  cells  in,  199 
starch-granules  in,  209 
sugars  in,  137 
sulphates    in,     111.       See    also 

Sulphates  in  urine. 
suppression,  88 
Taenia  echinococcus  in,  206 
tannin  in,  172 
thom-ajiple  cr\-stals  in,  187 
total  solids  in,  94 

Haser's  method,  96 
transparency,  90 
Trichomonas  vaginalis  in,  207 
tube-casts  in,  188 
tubercle  bacillus  in,  205 

detection,  205 
tyrosin  in,  180 

Momer's  test  for,  182 
urates  in  amorphous,  178 

in  mass,  197 
urea  in,  114.     See  also  Urea  in 

urine. 
urethral  shreds  in,  196 
uric  acid  in,  122.    See  also  Uri<: 

acid  in  urine. 
uric-acid  cr>'stals  in,  177 
urobiHn  in,  158 

Schlesinger's  test  for,  159 
urobilinogen  in,  158 

test  for,  159 


Urine,  urochloralic  acid  in,  167 

vinegar  eel  in,  207 

volatile  alkalinity,  92 

waxy  casts  in,  192 

yeast-cells  in,  208 
Urinometer,  Squibb's,  93 
Urinopyknometer,  Saxe's,  94,  95 
UrobiHn  in  urine,  158 

Schlesinger's  test  for,  159 
Urobilinogen  in  urine,  158 

test  for,  159 
Urochloralic  acicl  in  urine,  167 


Vaccines,  479 
autogenous,  479 
bottles,  480 
counting  of,  483 
dosage  of,  486 
in  infections,  487 
in  malignant  endocarditis,  487 
in  tv'phoid  fever,  488 
method  of  use,  485 
preparation  of,  479 
diluting,  485 

making  the  suspension,  481 
materials,  478 
obtaining  the  bacteria,  481 
sterilization,  482 
stock,  479 

therapeutic  indications,  486 
Vacuolated  leukocytes,  291 
Van  Gieson's  stain  for  Negri  bodies, 
Frothingham's  modification,  452 
Vegetable  cells  in  feces,  356 
fibers  in  feces,  356 
hairs  in  feces,  356 
Vernal  catarrh,  eosinophilic  leuko- 
cytes in,  440 
Vesical  calculus,  urine  in,  215 
tuberculosis,  urine  in,  215 
tumors,  urine  in,  215 
Vincent's  angina,  436 
spirochete  of,  374 
Vinegar  eel,  403 

in  urine,  207 
Viscosity  of  blood,  220 
Volatile  alkalinity  of  urine.  92 
Volhard's  estimation  of  chlorids  in 

urine,  104 
Volume  index  of  blood,  245 

Larrabee's  method,  246 


INDEX 


585 


Volume  index  of  blood,  method, 
245 

Von  Dungern's  test  for  malignant 
disease,  524 

Von  Fleischl's  estimation  of  hemo- 
globin, 226 
hemoglobinometer,   226 

Von  Jaksch's  anemia,  317 

Von  Pirquet's  reaction  in  tubercu- 
losis, 490 


Wassermann  reaction,  511 

amboceptor  in,  titration  of ,  516 
antisheep  amboceptor  in,  512 
complement  in,  513 

titration  of,  514 
errors  and  their  causes,  518 
interpretation  of  results,  522 
materials  required,  511 
patient's  serum  in,  513 
routine  methods,  522 
sheep's  red  blood-cells  in,  512 
syphilitic  antigen  in,  511 
titrations  in,  514 
with  cerebrospinal  fluid,  521 

Water-motor  centrifuge,  106 

Watery  blood,  219 

Waxy  casts  in  urine,  192 

Weights,  553 

Weil's  cobra-venom  test  for  sjrph- 
ilis,  528 

Weisz's  permanganate  test,  162 

WTiip-worm,  416 

White  blood-corpuscles,  218.     See 
also  Leukocytes. 

Whooping-cough  bacillus   in  spu- 
tum, 76 
lymphocytes  in,  280 
lymphocytic  leukocytosis  in,  253 

Widal  reaction,  496 

interpretation  of  results,  502 


Widal  reaction,  materials  required, 
498 
methods,  499 

macroscopic,  Bass  and  Wat- 
kins'  modification,  501 
microscopic,  499,  501 
obtaining  blood,  499 
Wires,  platiniun,  455 
Wool  fibers  in  urine,  197,  209 
Worms,  eosinophilia  as  symptom, 
286 
flat,  384 
guinea-,  408 
pin-,  40s 
roimd,  402 
screw,  418 
stomach,  405 
tape-,  384 
thread-,  405 
whip-,  416 
Wright  and  Kinnicutt's  estimation 

of  blood-plaques,  261 
Wright's  blood-stain  for  syphilis, 
448 
capsule,  457 
method  of  determining  opsonic 

index,  507 
stain  for  blood,  267 
for  malarial  parasites,  302 

Yaws,  Treponema  pertenue  of,  376 
Yeast-cells  in  gastric  contents,  341 

in  urine,  208 
Yeasts  in  sputiun,  61 


Zappert  ruling  for  count  in  leu- 
kemia, 253 

Ziehl-Neelson  method  for  Bacillus 
tuberculosis  in  sputiun,  65 

Zoomastigophora,  367,  372 


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fessor of  Surgery,  and  William  E.  Lower,  M.  D.,  Associate 
Professor  of  Geni to-urinary  Surgery,  Western  Reserve  University. 
Octavo  of  275  pages,  illustrated.     Cloth,  ^3.00  net. 

JUST  OUT— PRACTICAL  APPLICATION 

Anoci-association  robs  surgery  of  its  harshness,  diminishes  postoperative 
mortality,  lessens  postoperative  complications  (shock,  nausea,  vomiting,  gas 
pains,  backache,  nephritis,  pneumonia,  etc.).  You  get  here,  first  of  all,  a 
monograph  on  shock.  Then  follow  chapters  on  the  principles  of  rt«(7«'-rtj-j<>«a- 
tion;  the  technic  of  its  application  in  the  administration  of  the  anesthetic  in 
abdominal  operations,  in  gynecologic  operations,  in  genito-urinary  work,  in 
operations  for  cancer  of  the  breast,  rectum,  stomach,  uterus,  larynx,  and  tongue; 
in  exophthalmic  goiter  operations  ;  in  operations  on  brain  and  extremities. 

Crile  on  Emotions  just  out 

The  papers  in  this  volume  give  the  steps  by  which  Dr.  Crile  established  his 
kinetic  theory  of  shock.  They  show  the  j)hylogenetic  origin  of  the  emotions 
and  the  pathologic  identity  of  traumatic  and  emotional  shock,  and  point  out 
most  convincingly  the  clinical  application  of  these  premises  and  their  broad, 
general  significance  in  every  phase  of  life. 
Octavo  of  240  pages,  illustrated.     By  George  W.  Crile,  M.  D.     Cloth,  ^3  00  net. 


SAUNDERS'    BOOKS   ON 


Moynihan's  Abdominal  Operations 

Abdominal  Operations.  By  Sir  Berkeley  Moynihax,  M.  S.  (Lon- 
don), F.  R.  C.  S.,  of  Leeds,  England.  Two  octavos  of  500  pages  each, 
with  385  illustrations,  5  in  colors.      Per  set :  Cloth,  510.00  net. 

JUST  OUT— NEW  (3dj  EDITION,  RESET 

This  new  edition  has  been  issued  after  a  most  thorough  revision — so  thorough  that 
the  work  had  to  be- reset  and  issued  in  two  handsome  volumes.  Over  150  pages  of 
new  matter  and  80  new  illustiations  were  added.  Two  new  chapters  arc  those  on 
excision  of  gastric  ulcer  and  complete  gastrectomy.  Some  P5  illustrative  cases  are  dis- 
tributed throughout  the  work,  giving  every  detail,  history,  exam.ination,  operation, 
complications,  results.  These  are  extremely  instructive.  Moynihan's  definite, 
didactic  style,  together  with  the  large  nu"nber  of  practical  illustrations  and  the 
illustrative  cases  make  this  work  the  most  useful  abdominal  surgery  published. 
Each  volume  has  stamped  on  its  back  the  subjects  treated  therein,  thus  facilitating 
quick  consultation. 


Moynihan's  Duodenal  Ulcer  lotion 

Duodenal  Ulcer.  By  Sir  Berkeley  Moynihan,  M.  S.  (Lon- 
don), F.  R.  C.  S.,  of  Leeds,  England.  Octavo  of  486  pages,  illus- 
trated.    Cloth,  $5.00  net. 

"  Easily  the  best  work  on  the  subject  ;  coming,  as  it  does,  from  the  pen  of  one  of 
the  masters  of  surgery  of  the  upper  abdomen,  it  may  be  accepted  as  authoritative." 
— The  London  Lancet. 


Moynihan  on  Gall-stones  t^Zt 

G.\LL-STONES  AND  ThEIR  SuRGICAL  TREATMENT.     By  SiR  BERKELEY 

Moynihan,  M.  S.  (London),  F.  R.  C.  S.    Octavo  of  458  pages,  illus- 
trated.    Cloth,  $5.00  net. 

"  He  expresses  his  views  with  admirable  clearness,  and  he  supports  them  by  a 
large  number  of  clinical  examples,  which  will  be  much  prized  by  those  who  know 
the  difficult  problems  and  tasks  which  gall-stone  surgery  not  infrequently  pre.-ients." 
— British  Medical  Journal. 


SURGEH  Y  AND    ANA  lOMY. 


Hornsby  and  ScKmidt's 
The  Modem  Hospitdwl 

The  Modern  Hospital.  Its  Inspiration  ;  Its  Construction ; 
Its  Equipment ;  Its  Management.  By  John  A.  Hornsby,  M.D., 
Secretary,  Hospital  Section,  American  Medical  Association ;  and 
Richard  E.  SchiMidt,  Architect.  Large  octavo  of  644  pages, 
with  207  illustrations.    Cloth,  $7.00  net ;  Half  Morocco,  ^8.50  net. 

ADOPTED  AT  ONCE  BY  THE  U.  S.  GOVERNMENT  AS  "THE  LAW" 

"Hornsby  and  Schmidt"  tells  you  just  exactly  how  to  plan,  construct, 
equip,  and  manage  a  hospital  in  all  its  departments,  giving  you  every  detail. 
It  gives  you  exact  data  regarding  heating,  ventilating,  plumbing,  refrigerating, 
etc. — and  the  costs.  It  tells  you  how  to  equip  a  modern  hospital  with  modern 
appliances.  It  tells  you  what  you  need  in  the  operating  room,  the  wards,  the 
private  rooms,  the  dining  room,  the  kitchen — every  division  of  hospital  house- 
keeping. It  gives  you  definite  diets  for  the  patients  and  the  hospital  house- 
hold. It  gives  you  hundreds  of  valuable  points  on  the  business  management 
of  hospitals — large  and  small. 


Allen's  Local  Anesthesia 

Local  Anesthesia.  By  Carroll  W.  Allen,  M,  D.,  In- 
structor in  Clinical  Surgery  at  Tulane  University  of  Louisiana. 
Octavo  of  625  pages,  illustrated.     Cloth,  ;$6.oo  net. 

JUST  READY 

This  is  a  complete  work  on  this  subject.  You  get  the  history  of  local 
anesthesia,  a  chapter  on  nerves  and  sensation,  giving  particular  attention  to 
pain — what  it  is  arid  its  psychic  control.  Then  comes  a  chapter  on  osmosis 
and  diffusion.  Eacn  local  anesthetic  is  taken  up  in  detail,  giving  very  special 
attention  to  cocain  and  novocain,  pointing  out  the  action  on  the  nervous  system, 
the  value  of  adrenalin,  paralysis  caused  by  cocain  anesthesia,  control  of  tox- 
icity. You  get  Crile's  method  of  administering  adrenalin  and  salt  solution, 
the  exact  way  to  produce  the  intradermal  wheal,  to  pinch  the  flesh  for  the  inser- 
tion of  the  needle — all  shown  you  step  by  step.  You  get  an  article  on  anoci- 
associaiioHy  the  production  of  local  anesthesia  in  the  various  regions,  spinal 
analgesia,  and  epidural  injections.    There  is  a  large  section  on  dental  anesthesia. 


SAUNDERS'    BOOKS    ON 


Cotton's 
Dislocations  and  Joint  Fractures 


Dislocations  and  Joint  Fractures.  By  Frederic  Jay 
Cotton,  A.  M.,  M.  D.,  First  Assistant  Surgeon  to  the  Boston 
City  Hospital.  Octavo  volume  of  654  pages,  with  1201  original 
illustrations.     Cloth,  $6.00  net. 

TWO  PRINTINGS  IN  EIGHT  MONTHS 

Dr.  Cotton's  clinical  and  teaching  experience  in  this  field  has  especially 
fitted  iiim  to  write  a  piactical  work  on  this  subject.  He  has  written  a  book 
clear  and  definite  in  style,  systematic  in  presentation,  and  accurate  in  state- 
ment. The  author  is  himself  tiie  artist,  so  that  the  illustrations  show  just 
those  points  he  wi>hed  to  emphasize. 

Boston  Medical  and  Surgical  Journal 

"The  work  is  delightful,  spirited,  scholarly,  and  original.  It  brings  the  subjects  up 
to  date — a  feat  long  neglected." 


Murphy's  Famous  Clinics 

Surgical  Clinics  of  John  B.  Murphy,  M.  D.,  at  Mercy 
Hospital,  Chicago.  Issued  serially,  one  number  every  other 
month  !  si.\  numbers  a  year).  Each  issue  about  200  octavo  pages, 
illustrated.  Per  year:  $8.00  ;  Cloth,  ^12.00.  Sold  only  by  the 
calendar  year. 

YOU  NOW  GET  DR.  MURPHY'S  METHODS  OF  DIAGNOSIS 

This  is  just  the  work  you  have  been  waitin<j  for — a  permanent  record  of 
the  teachings  of  this  great  surgeon.  These  Clinics  are  publisheti/wj/  as  de- 
livered by  Dr.  Murphy,  an  expert  stenographer  taking  down  everything  Dr. 
Murphy  says  and  does.  In  this  way  these  Cl.lMCS  retain  all  that  individual 
force  and  charm  so  characteristic  of  the  clinical  teaching  of  this  distinguished 
surgeon.  But  the  most  vital  point  about  these  Cl.iNlCS  is  that  they  are  abso- 
btlely  fresh.  They  are  surgery — practical,  applied  surgery — right  down  to  the 
minute,  published  practically  as  soon  as  delivered.  There  is  no  stale  matter. 
Everything  is  new,  never  having  appeared  in  print  before.  It  is  all /iV^ 
clinical  material. 


SURGERY  AND  ANATOMY 


Crandon  and  Ehrenfried's 
Surgical    After  -treatment 

Surgical  After-treatment.  By  L.  R.  G.  Crandon,  M.  D., 
Assistant  in  Surgery,  and  Albert  Ehrenfried,  M.  D.,  Assistant 
in  Anatomy,  Harvard  Medical  School.  Octavo  of  831  pages, 
with  265  original  illustrations.  Cloth,  ^6.00  net. 

THE  NEW  (2d)  EDITION 

This  worK  tells  how  best  to  manage  all  problems  and  emei^encies  of  sur- 
gical  convalescence  from  recovery-room  to  discharge.  It  gives  all  the  detailt 
completely,  definitely,  yet  concisely,  and  does  not  refer  the  reader  to  some 
other  work  perhaps  not  then  available.  The  postoperative  conduct  of  all 
operations  is  given.  There  is  an  elaborate  chapter  pn  Vaccine  Therapy,  Ini' 
munizalion  by  Inoculation,  and  Specific  Sera,  by  Dr.  Geoi^e  P.  Sanborn. 
Therapeutic  Gazette 

"  This  book  is  one  which  can  be  read  with  profit  by  the  active  surgeon  and  practitioner 
and  will  be  generally  commended." 

Collected  Papers  by  the  Staff  of  St. 
Mary's  Hospital,  Mayo  Clinic 

Collected  Papers  by  the  Staff  of  St.  Mary's  Hospital, 
Mayo  Clinic.  By  William  J.  Mayo,  M.D.,  Charles  H. 
Mayo,  M.D.,  and  their  Associates  at  St.  Mary's  Hospital, 
Rochester,  Minn.  Papers  of  1905-09,  1910,  1911,  1912,  1913, 
1914.     Each,  $5.50  net. 


A  Collection  of  Papers  (published  previous  to  1909).  By 
W.  J.  and  C.  H.  Mayo.  Two  octavos  of  525  pages  each,  illus- 
trated.    Per  set:  Cloth,  ;^i 0.00  net. 


SAUNDERS'  BOOKS  ON 


Keen's  New  Surgery 

Surgery :  Its  Principles  and  Practice.  Written  by  82 
ominent  specialists.  Edited  by  W.  W.  Keen,  M.  D.,  LL.  D., 
Hon.  F.  R.  C.  S.,  Eng.  and  Edin.,  Emeritus  Professor  of  the 
Principles  of  Surgery  and  of  Clinical  Surgery  at  the  Jefferson 
Medical  College,  Philadelphia.  Six  large  octavo  volumes  of 
over  1050  pages  each,  containing  3100  illustrations,  157  in  colors. 
Per  volume  :   Cloth,  $7.00  net;  Half  Morocco,  $8.00  net. 

VOLUME  VI  GIVES  YOU  THE  NEWEST  SURGERY 

In  this  sixth  volume  you  get  all  the  newest  surgery — both  general  and 
special — from  the  pens  of  those  same  international  authorities  who  have  made 
the  success  of  Keen's  Surgery  world-wide.  Each  man  has  searched  for  the 
new,  the  really  useful,  in  his  particular  field,  and  he  gives  it  to  you  here. 
Here  you  get  the  newest  surgery,  and  fully  illustrated.  Then,  further,  you 
get  a  complete  index  to  the  entire  six  volumes,  covering  125  pages,  but  so 
arranged  that  reference  to  it  is  extremely  easy.  If  you  want  the  newest  sur- 
gery, you  must  turn  to  the  new  "  Keen  "  for  it. 


Bryan's   Surg'ery 

Principles  of  Surgery.    By  W.  A.  Bryan,  M.D.,  Professor 

of  Surgery  and  Clinical  Surgery  at  Vanderbilt  University,  Nash- 
ville. Octavo  of  677  pages,  with  224  original  illustrations. 
Cloth,  $4.00  net.  • 

MANY  NEW  IDEAS 

Dr.   Bryan  here  gives  you  facts,  accurately  and  concisely  stated,  without  , 
which  no  modern  practitioner  can  do  modem  work.     He  discredits  many 
fallacious  ideas,  giving  yow  fads  instead.     He  shows  you  in  a  most  practical 
way  the  relations  between  surgical  pathology  and  the  resulant  symptomatology, 
and  points  out  the  influence  such  information  has  on  treatment. 

Dr.  A.  Vander  Veer,  Albany  Medical  College 

"  It  comes  to  us  full  of  new  ideas.     So  much  that  is  clear  and  concise  has  been  added 
that  it  is  fascinating  to  study  the  work.     It  is  bound  to  receive  a  hearty  welcome." 


SURGER  Y  AND  ANA  TO  MY 


Scudder's  Fractures 

WITH    NOTES   ON   DISLOCATIONS 


The  Treatment  of  Fractures  :  with  Notes  on  a  few  Com- 
mon Dislocations,  By  Charles  L.  Scudder,  M.  D.,  Surgeon  to 
the  Massachusetts  General  Hospital,  Boston.  Octavo  volume  of 
708  pages,  with  994  illustrations.      Polished  Buckram,  $6.00  net. 

THE  NEW  (7th)  EDITION,  ENLARGED 

OVER  33.500  COPIES 

Seven  large  editions  of  this  remarkable  book  is  a  decisive  indication 
of  the  value  of  Dr.  Scudder' s  work.  For  this  new  edition  numerous  ad- 
ditions have  been  made  throughout  the  text  and  a  large  number  of  new 
illustrations  added,  greatly  enhancing  the  value  of  the  work.  In  every  way 
this  edition  reflects  the  very  latest  advances  in  the  treatment  of  fractures. 
J.  F,  Binnie,  M.  D.,  formerly  University  of  Kansas. 

"  Scudder's  Fractures  is  the  most  successfnl  Look  on  the  subject  that  has  ever  been 
published.     I  keep  it  at  hand  regularly." 


Scudder's  Tumors  of  the  Jaws 

Tumors  of  the  Jaws.  By  Charles  L.  Scudder,  M.  D., 
Surgeon  to  the  Massachusetts  General  Hospital,  Boston.  Octavo 
of  395  pages,  with  353  illustrations,  6  in  colors.     Cloth,  ^6.00  net. 

WITH  NEW  ILLUSTRATIONS 

Dr.  Scudder  in  this  book  tells  you  how  to  determine  in  each  case  the 
form  of  new  growth  present,  and  then  points  out  the  best  treatment.  As  the 
tendency  of  malignant  disease  of  the  jaws  is  to  grow  into  the  accessory  sinuses 
and  toward  the  base  of  the  skull,  an  intimate  knowledge  of  the  anatomy  of 
these  sinuses  is  essential.  Dr.  Scudder  has  included,  therefore,  sufficient 
anatomy  and  a  number  of  illustrations  of  an  anatomic  nature.  Whether  gen- 
eral practitioner  or  surgeon,  you  need  this  new  book  because  it  gives  you  just 
the  information  you  want. 


SAUNDERS'    BOOKS   ON 


Sisson's 
Anatomy  of  Domestic  Animals 


Anatomy  of  Domestic  Animals.  By  Septimus  Sisson, 
S.  B.,  V.  S.,  Professor  of  Comparative  Anatomy  in  Ohio  State 
University.  Octavo  volume  of  930  pages,  with  726  illustrations, 
mostly  original  and  many  in  colors.     Cloth,  $7.00  net. 

JUST  OUT— NEW  (2d)  EDITION.  RESET 

This  is  a  clear  and  concise  statement  of  the  essential  facts  regarding  the 
structure  of  the  principal  domesticated  animals,  containing  many  hitherto 
unpublished  data  resulting  from  the  detailed  study  of  formalin-hardened 
subjects  and  frozen  sections.  The  author  has  devoted  much  time  and  car"; 
to  the  illustrations.  This  is  the  only  veterinary  anatomy  in  thirty  years.  The 
new  nomenclature  is  used. 

Boston  Medic&I  and  Surreal  Journal 

"This  fine  piece  of  bookmaking  is  comparable  to  the  best  works  on  human  anatomy, 
and  is  a  decided  addition  to  good  veterijiary  li-erature." 


Lynch  on  Diseases  of  Swine 

Diseases  of  Swine.  With  particular  reference  to  Hog 
Cholera.      By  Charles   F.   Lynch,  M.  D.,  D.  V.  S.,  Terra 

Haute  Veterinary  College.  Chapter  on  Castration  and  Spaying 
by  George  R.  White,  M.  D.,  D.  V.  S.,  State  Veterinarian  of 
Tennessee.     Octavo  of  741  pages,  illustrated.     Cloth,  $5.00  net. 

OVER  400  PAGES  ON  HOG  CHOLERA 

This  is  one  of  the  most  important  contributions  to  veterinary  literature 
published  for  some  time.  You  get  first  some  80  pages  on  the  various  breeds 
of  hogs,  with  valuable  points  in  jtuiging  swine.  Then  comes  an  extremely 
important  monograph  of  over  400  pages  on  hog  cholera,  giving  the  history, 
causes,  pathology,  types,  and  treatment.  On  hog-cholera  serum  alone  you 
get  ot'er  200  pages,  telling  you  how  to  manufacture  the  serum,  how  to  use  it, 
when  to  use  it,  the  prophylactic  dose,  handling  of  complications,  everything 
on  this  question  of  hog-cholera  serum.  Then,  in  addition,  you  get  complete 
chapters  on  all  other  diseases  of  swine,  both  medical  and  surgical,  and  a 
chapter  on  castration  and  spaying. 


SURGERY  AND  ANATOMY 


Kelly  and  Noble's  Gynecology 
and  Abdominal  Sur£(ery 

Gynecology  and  Abdominal  Surgery.  Edited  by  Howard 
A.  Kelly,  M.  D.,  Professor  of  Gynecology  in  Johns  Hopkins 
University;  and  Charles  P.  Noble,  M.  D.,  formerly  Clinical 
Professor  of  Gynecology,  Woman's  Medical  College,  Philadel- 
phia. Two  imperial  octavos  of  950  pages  each,  with  880  illustra- 
tions.    Per  volume :  Cloth,  $8.00  net ;  Half  Morocco,  1^9.50  net. 

WITH  880  ILLUSTRATIONS  BY  BECKER  AND  BRODEL 

This  work  possesses  a  number  of  valuable  features  not  to  be  found  in  any- 
other  publication  covering  the  same  fields.  It  contains  a  chapter  upon  the 
bacteriology  and  one  upon  the  pathology  of  gynecology,  and  a  large  chapter 
devoted  entirely  to  medical  gynecology ,  written  especially  for  the  physician 
engaged  in  general  practice.  Abdominal  surgery  proper,  as  distinct  from 
gynecology,  is  fully  treated,  embracing  operations  upon  the  stomach,  intes- 
tines, liver,  bile-ducts,  pancreas,  spleen,  kidneys,  ureter,  bladder,  and  peri- 
toneum. 

American  Journal  of  Medical  Sciences 

"It  is  needless  to  say  that  the  work  has  been  thoroughly  done ;  the  names  of  the 
authors  and  editors  would  guarantee  this,  but  much  may  be  said  in  praise  of  the  method 
of  presentation ;  and  attention  may  be  called  to  the  inclusion  of  matter  not  to  be  found 
elsewhere." 


Bickham's  Operative  Surgery 

A  Text-Book  of  Operative  Surgery.  By  Warren  Stone 
BiCKHAM,  M.  D.,  of  New  York.  Octavo  of  1200  pages,  with 
854  original  illustrations.  Cloth,  $6.50  net;  Half  Morocco, 
$8.00  net. 

THIRD  EDITION 

Boston  Medical  toid  Surgical  Journal 

"  The  book  is  a  valuable  contribution  to  the  literature  of  operative  surgery.  It  repre" 
sents  a  vast  amount  of  careful  work  and  technical  knowledge  on  the  p  art  of  the  author- 
For  the  surgeon  in  active  practice  or  the  instructor  of  surgery  it  is  an  unusually  good  review 
of  the  subject." 


SAUNDERS'    BOOKS   ON 


Mumford's 
Practice   of  Surgery 

The  Practice  of  Surgery.  By  James  G.  Mumford,  M,  D., 
Associate  in  Surgery,  Harvard  Medical  School.  Octavo  of  1032 
pages,  with  683  illustrations.     Cloth,  $7.00  net. 

JUST  OUT— NEW   (2d)  EDITION 

This  is  a  clinical  suri^ery,  giving  those  methods  and  operations  which  the 
author  has  personally  followed  for  the  past  twenty  years.  The  plan  of  the 
work  is  somewhat  off  the  conventional  lines,  the  diseases  being  taken  up  in 
their  order  of  interest,  importance,  and  frequency. 

John    B.    Murphy,    M.  D.,   Northwestern  Medical  School,   Chicago 

"This  work  truly  represents  Dr.  Mumford's  intellectual  capacity  and  scope,  and  pre- 
sents in  a  terse,  forceful,  yet  pleasing  manner,  the  live  surgical  topics  of  the  day.  It  is  in 
every  particular  up  to  date." 

DaCosta's  Modern  Surgery 

Modern  Surgery — General  and  Operative.  By  John 
Chalivjers  DaCosta,  M.  D.,  Samuel  D.  Gross  Professor  of  Sur- 
gery, Jefferson  Medical  College,  Philadelphia.  Octavo  of  15 15 
pages,  with  1085  illustrations.  Cloth,  $6.00  net;  Half  Morocco, 
^7.50  net. 

NEW  (7th)    EDITION 

A  surgery,  to  be  of  the  maximum  value,  must  be  up  to  date,  must  be  com- 
plete, must  have  behind  its  statements  the  sure  authority  of  experience,  must 
be  so  arranged  that  it  can  be  consulted  quickly ;  in  a  word,  it  must  be  practical 
and  dependable.  Such  a  surgery  is  DaCosta's.  Always  an  excellent  work 
for  this  edition  it  has  been  very  materially  improved  by  the  addition  of  much 
new  matter  and  many  additional  illustrations. 

Rudolph  M&tas,  M.D.,  Professor  of  Surgery,  Tulane  University  of  Louisiana. 

"  This  edition  is  destined  to  rank  as  high  as  its  predecessors,  which  have  placed  the 
learned  author  in  the  fore  of  text-book  writers.  The  more  I  scrutinize  its  pages  the  more  I 
admire  the  marvelous  capacity  of  the  author  to  compress  so  much  knowledge  in  so  small  a 
space." 


SURGERY  AND  ANATOMY  \x 

Feng(er  Memorial  Volumes 

Collected  Works  of  Christian  Fenger,  M.  D.  Edited  by 
LuDviG  Hektoen,  M.  D.,  Professor  ot  Pathology,  Rush  Medical 
College,  Chicago.  Two  octavos  of  525  pages  each.  Per  set: 
Cloth,  $15.00  net;  Half  Morocco,  ;^i8.oo  net. 

LIMITED  EDITION 

These  handsome  volumes  consist  of  all  the  important  papers  written  by  the 
late  Christian  Fenger.  Not  only  the  papers  published  in  English  are  in- 
cluded, but  also  the  translations  of  those  which  originally  appeared  in  Danish, 
German,  and  French. 


Sobotta  and  McMurrich's 
Human  Anatomy 

Atlas   and   Text- Book   of  Human  Anatomy.     In  Three 

Volumes.     By  J.  Sobotta,  M.  D.,  of  Wiirzburg.     Edited,  with 

additions,  by  J.  Playfair  McMurrich,  A.  M.,  Ph.  D.,  Professor 

of  Anatomy,  University  of  Michigan.     Three  large  quartos,  each 

containing  250  pages  of  text  and  over  300  illustrations,  mostly  in 

colors.  Per  volume  :  Cloth,  ^6.00  net. 

Edward  Martin,  M.D.,  University  of  Pennsylvania. 

"  This  is  a  piece  of  bookmaking  which  is  truly  admirable,  with  plates  and  text  so  well 
chosen  and  so  clear  that  the  work  is  most  useful." 

Campbell's  Sur££ical  Anatomy  |5^;J 

A  Text-Book  of  Surgical  ANATO>nr.  By  William  Francis 
Campbell,  M.  D.,  Professor  of  Anatomy,  Long  Island  College  Hospital. 
Octavo  of  675  pages,  with  319  original  illustrations.     Cloth,  JS5.00  net 

This  is  in  the  fullest  sense  an  applied  anatomy — an  anatomy  that  will  be  of  inesti- 
mable value  to  the  surgeon  because  only  those  facts  are  discussed  and  only  tboce 
structures  and  regions  emphasized  that  have  a  peculiar  interest  to  him. 


SAUNDERS^  BOOKS  ON 


Eisendrath's 
Surgical  Diagnosis 

A  Text-Book  of  Surgical  Diagnosis.  By  Daniel  N.  Eisen- 
DRATH,  M.  D.,  Professor  of  Surgery  in  the  College  of  Physicians 
and  Surgeons,  Chicago.  Octavo  of  885  pages,  with  574  entirely 
new  and  original  text-illustrations  and  some  colored  plates. 
Cloth,  $6.50  net;   Half  Morocco,  $8.00  net. 

SECOND  EDITION 
WITH    574    ORIGINAL    ILLUSTRATIONS 

Dr.  Eisendrath  takes  up  each  disease  and  injury  amenable  to  surgical 
treatment,  and  sets  forth  the  means  of  correct  diagnosis  in  a  systematic  and 
comprehensive  way.  Definite  directions  as  to  methods  of  examination  are  pre- 
sented clearly  and  concisely,  providing  for  all  contingencies  that  might  arise 
in  any  given  case.  Each  one  of  the  four  hundred  and  eighty-two  magnifi- 
cent illustrations  indicates  precisely  how  to  diagnose  the  condition  considered. 

Surgery,  Gynecolo^,  zmd  Obstetrics 

"  The  book  is  one  which  is  well  adapted  to  the  uses  of  the  practising  surgeon  who 
desires  information  concisely  and  accurately  given." 

Eisendrath's  Clinical  Anatomy 

A  Text-Book  of  Clinical  Anatomy.  By  Daniel  N.  Eisen- 
drath, A.  B.,  M.  D.,  Professor  of  Surgery  in  the  College  of 
Physicians  and  Surgeons,  Chicago.  Octavo  of  535  pages,  with 
original  illustrations.     Cloth,  $5.00  net. 

SECOND  EDITION 

This  new  anatomy  discusses  the  subject  from  the  clinical  standpoint.     A 
portion  of  each  chapter  is  devoted  to  the  examination  of  the  living  through 
palpation  and  marking  of  surface  outline  of  landmarks,  etc.     The  illustrations 
are  original. 
Medical  Record,  New  York 

"  A  special  recommendation  for  the  figures  is  that  they  are  mostly  original  and  were 
made  for  the  purpose  in  view.  The  sections  of  joints  and  trunks  are  those  of  formalinized 
cadavers  and  are  unimpeachable  in  accuracy." 


SURGERY  AND  ANATOMY  13 

Gould's  Operations  on  Intestines  and  Stomach 

The  Technicof  Operations  Upon  the  Intestines  and  Stomach. 
By  Alfred  H.  Gould,  M.D.,  of  Boston.  Lai^e  octavo,  with  190 
original  illustrations,  some  in  colors.  Cloth,  I5.00  net ;  Half  Morocco, 
$6.50  net. 

"  The  illustrations  are  so  good  that  one  scarcely  needs  the  text  to  elucidate  the  steps 
of  the  operations  described.  The  work  represents  the  best  surgical  knowledge  and 
skill." — Neiu  York  State  Journal  0/ Mediciru. 

Dannreuther's  Emergency  Surgery 

Minor  and  Emergency  Surgery.  By  Walter  T.  Dannreuther, 
M.  D.,  Surgeon  to  St.  Elizabeth's  Hospital  and  to  St.  Bartholomew's 
Clinic,  New  York  City.  l2mo  of  225  pages,  illustrated.  Cloth,  $1.25 
net. 

Griffith's  Hand-Book  of  Surgery 

A  Manual  of  Surgery.  By  Frederic  R.  Griffith,  M.  D.,  Sur- 
geon to  the  Bellevue  Dispensary,  New  York  City.  l2mo  of  579  pages, 
with  417  illustrations.     Flexible  leather,  ;$2.oo  net 

Keen's  Addresses  and  Other  Papers 

'  Addressf.s  and  Other  Papers.  Delivered  by  William  W.  Keen, 
M.  D.,  LL.D..  F.  R.  C.  S.  (Hon.),  Professor  of  the  Principles  of  Surgery 
and  of  Clinical  Surgery.  Jefferson  Medical  College,  Philadelj  hia.  Octavo 
volume  of  441  pages,  illustrated.     Cloth,  $3-75  "^t- 

Keen  on  the  Surgery  of  Typhoid 

The  Surgical  Complications  and  Sequels  of  Typhoid  Fever. 
By  Wm.  W.  Keen,  M.  D.,  LL.D.,  F.  R.  C.  S.  (Hon.),  Professor  of 
the  Principles  of  Surgery  and  of  Clinical  Surgery,  Jefferson  Medical 
College,  Philadelphia,  etc.  Octavo  volume  of  386  pages,  illustrated. 
Cloth,  ;$3.oo  net. 

American  Text-Book  of  Surgery  Fourth  Edition 

American  Text- Book  of  Surgery.  Edited  by  W.  W.  Keen,  M.  D., 
LL.  D.,  Hon.  F.  R.  C.  S.,  Eng.  and  Edin.;  and  J.  William  White, 
M.  D.,  Ph.  D.  Octavo,  1363  pages,  551  text-cuts  and  39  colored  and 
half-tone  plates.     Cloth,  57-00  net ;   Half  Morocco,  $8.50  net. 

Robson  and  Cammidge  on  the  Pancreas 

The  Pancreas  :  Its  Surgery  and  Pathology.  By  A.  W.  Mayo 

Robson,    F.  R.  C.  S.,    of  London,    England  ;    and  P.  J.  Cammidge, 

F.  R.  C.  S.,  of  London,  England.  Octavo  of  546  pages,  illustrated. 
Cloth,  $5.00  net;  Half  Morocco,  $6.50  net. 


14  SAUA'DERS'    BOOKS  ON 

American  Illustrated  Dictionary   ^^^  (7^^)  Edition 

The  American  Illustrated  Medical  Dictionary.  With 
tables  of  Arteries,  Muscles,  Nerves,  Veins,  etc.  ;  of  Bacilli, 
Bacteria,  etc.  ;  Eponymic  Tables  of  Diseases,  Operations, 
Stains,  Tests,  etc.  By  W.  A.  NE\v^L•^LN  Dorland,  M.  D. 
Large  octavo,  1107  pages.  Flexible  leather,  $4.50  net ;  with 
thumb  index,  $5.00  net. 

Howard    A.    Kelly,  M.  D.,    Professor  of   Gynecology,  Johns  Hopkins 
"Dr.  Dorland  s  dictionary  is  admirable.     It  is  so  well  gotten  up  and  of 
such  convenient  size.     No  errors  have  been  found  in  my  use  of  it." 

Golebiewski  and  Bailey's  Accident  Diseases 

Atlas  and  Epitome  of  Diseases  Caused  by  Accidents. 
By  Dk.  Ed.  Golebiewski,  of  Berlin.  Edited,  with  additions, 
by  Pearce  Bailey,  M.D.  Cloth,  I4.00  net.  ///  Saunders' 
Hand- Atlas  Series. 

Helferich  and  Bloodgood  on  Fractures 

Atlas  and  Epitome  of  Traumatic  Fractures  and  Dislo- 
cations. By  Prof.  Dr.  H.  Helferich,  of  Greifswald,  Prussia. 
Edited,  with  additions,  by  Joseph  C.  Bloodgood,  M.D.,  Asso- 
ciate in  Surgery,  Johns  Hopkins  University,  Baltimore.  .216 
colored  figures  on  64  lithographic  plates,  190  text-cuts,  and 
353  pages  of  text.   Cloth,  $3.00  net.  ///  Sounders'  Atlas  Series. 

Sultan  and  Coley  on  Abdominal  Hernias 

Atlas  and  Epitome  of  Abdominal  Hernias.  By  Pr.  Dr. 
G.  Sultan,  of  Gottingen.  Edited,  with  additions,  by  Wm. 
B.  Coley,  M.D.  Cloth,  $3. 00  net.  In  Saunders'  Hand- Atlas 
Series. 

Warren's  Surgical  Pathology  ^^ 

Surgical  Pathology  and  Therapeutics.  By  J.  Collins 
Warren,  M.D.,  LL.D.,  F.R.C.S.(Hon.),  Professor  of  Sur- 
gery, Harvard  Medi^^al  School.  Octavo,  873  pages,  136  illus- 
trations.    Cloth,  $5.00  net ;  Half  Morocco,  $6.50  net. 

Zuckerkandl  and  DaCosta's  Surgery  f^jj 

Atlas  and  Epitome  of  Operative  Surgery.  By  Dr.  O. 
Zuckerkandl,  of  Vienna.  Edited,  with  additions,  by  J. 
Chalmers  DaCosta,  M.  D.,  Samuel  D.  Gross  Professor  of 
Surgery,  Jefferson  Medical  College,  Philadelphia.  40  col- 
ored plates,  278  text-cuts,  and  410  pages  of  text.  Cloth, 
$3.50  net.     In  Saunders'  Atlas  Series. 


SURGER  Y  AND  ANA  TO  MY-  15 

Schultze  and  Stewart's  Topographic  Anatomy 

Atlas  and  Text-Book  of  Topographic  and  Applied  Anatomy. 
By  Prof.  Dr.  O.  Schultze,  of  Wurzburg.  Edited,  with  additions,  by 
George  D.  Stewart,  M.  D.,  Professor  of  Anatomy  and  Clinical  Sur- 
gery, University  and  Bellevue  Hospital  Medical  College,  N.  Y.  La^e 
quarto  of  189  pages,  with  25  colored  figures  on  22  colored  lithographic 
plates,  and  89  text-cuts,  60  in  colors.     Cloth,  $5.50  net. 

"J  regard  Schultze  and  Stewart's  Topographic  and  Applied  Anatomy  as  a  very 
admirable  work,  for  students  especially,  and  I  find  the  plates  and  the  text  excel- 
lent."— Arthur  Dean  Sevan,  M.  D.,  Professor  of  Surgery  in  Rush  Medical  Col' 
lege,  Chicago. 

Metheny's  Dissection  Methods 

Dissection  Methods  and  Guide.  Octavo  of  131  pages,  illustrated. 
By  David  Gregg  Metheny,  M.  D.,  L.  R.  C.  P.,  L.  R.  C.  S.  (Edin.), 
L.  F.  P.  S.  (Glas.  ),  Associate  in  Anatomy  at  Jefferson  Medical  College, 
Philadelphia.     Cloth,  if  1.25  net. 

American  Pocket  Dictionary         ^ew  (sth)  Edition 

The  American  Pocket  Medical  Dictionary.  Edited  byW.  A. 
Newman  Dorland,  A.M.,  M.D.  677  pages.  Full  leather,  limp,  with 
gold  edges,  $1.00  net;  with  patent  thumb  index,  ;j5i.25  net. 

Barton  and  Wells'  Medical  Thesaurus 

A  Thesaurus  of  Medical  Words  and  Phrases.  ByW.  M.  Barton, 
A.  M.,  M.  D.,  Assistant  to  Professor  of  Materia  Medica  and  Therapeutics, 
Georgetown  University,  Washington,  D.  C.  ;  and  Walter  A.  Wells, 
M.  D.,  Demonstrator  of  Laryngology,  Georgetown  University,  Washing- 
ton, D.  C.  i2mo  of  534  pages.  Flexible  leather,  ;^2.5o  net;  thumb 
index,  jf3.oo  net. 

Meyer  &  Schmieden's  Bier's  Hyperemic  Treatment 

Second  Edition 

Bier's  Hyperemic  Treatment  in  Surgery,  Medicine,  and  the  Special- 
ties. By  Willy  Meyer,  M.  D.,  Professor  of  Surgery,  New  York  Post- 
Graduate  Medical  School  and  Hospital;  and  Prof.  Dr.  Victor  Schmie- 
DEN,  Assistant  to  Professor  Bier,  University  of  Berlin,  Germany.  Octavo 
of  280  pages,  illustrated.     Cloth,  ;P3.oo  net. 

Morris*  Dawn  of  the  Fourth  Era  in  Surgery 

Dawn  of  the  Fourth  Era  in  Surgery  and  Other  Articles. 
By  Robert  T.  Morris,  M.  D.,  Professor  of  Surgery,  New  York  Post- 
Graduate  Medical  School  and  Hospital.  I2mo  of  145  pages,  illustrated. 
51.25  net. 


I6  SURGER  Y  AND  ANA  TOMY 

Moore's  Orthopedic  Surgery 

A  Manual  of  Orthopedic  Surgery.  By  James  E.  Moore,  M.  D,, 
Professor  of  Clinical  Surgery,  University  of  Minnesota,  College  of  Medi- 
cine and  Surgery.  Octavo  of  356  pages,  handsomely  illustrated.  Cloth, 
I2.50  net. 

Fowler's  Operating  Room  New  (3d)  E:ditioii 

The  Operating  Room  and  the  Patient.  By  Russell  S.  Fowler, 
M.  D.,  Chief  Surgeon,  First  Division,  German  Hospital,  Brooklyn,  New 
York.     Octavo  of  611  pages,  illustrated.     Cloth,  J3.50  net. 

International  Text-Book  of  Surgery         Second  Edition 

The  International  Text-Book  of  Surcery.  In  two  volumes. 
By  American  and  British  authors.  Edited  by  J.  CoLi.lNS  Warren, 
M.  D.,  LL.  D.,  F.  R.  C.  S.  (Hon.),  Professor  of  Surgery,  Harvard 
Medical  School;  and  A.  Pearce  Gould,  M.  S.,  F.  R.  C.  S.,  of 
London,  England.  Vol.  I.  :  General  and  Operative  Sitrs^eiy.  Royal 
octavo,  975  pages,  461  illustrations,  9  full-page  colored  plates.  Vol. 
II.:  Special  or  Rei^ional  Sun^ery.  Royal  octavo,  1 122  pages,  499 
illustrations,  and  8  full-page  colored  plates.  Per  volume :  Cloth,  ^^5. CO 
net;  Half  Morocco,  $6.50  net. 

Nancrede's  Principles  of  Surgery  Second  EUlition 

Lectures  on  the  Principles  of  Surgery.  By  Chari.es  B.  Nan- 
crede,  M.  D.,  LL.  D.,  Professor  of  Surgery  and  of  Clinical  Surgery. 
University  of  Michigan,  Ann  Arbor.  Octavo,  407  pages,  illustrated. 
Cloth,  $2.50  net. 

Nancrede's  Essentials  of  Anatomy.  7th  Edition 

Essentials  of  Anatomy,  including  the  Anaiomy  of  the  Viscera. 
By  Charles  B.  Nancrede,  M.  D.,  Professor  of  Surgery  and  of  Clinical 
Surgery,  University  of  Michigan,  Ann  Arbor.  Crown  octavo,  388  pages, 
180  cuts.  With  an  Appendix  containing  over  60  illustrations  of  the 
osteology  of  the  body.  Based  on  Grafs  Anatomy.  Cloth,  ^l.oo  net. 
In  Saunders'  Question  Compends. 

Martin's  Essentials  of  Surgery.    Seventii  Revised  Edition 

Essentials  of  Surgery.  Containing  also  Venereal  Diseases,  Surgi- 
cal Landmarks,  Minor  and  Operative  Surgery,  and  a  complete  description, 
with  illustrations,  of  the  Handkerchief  and  Roller  Bandages.  By  Ed- 
ward Martin,  A.  M.,  M.  D.,  Professor  of  Clinical  Surgery,  University 
of  Pennsylvania,  etc.  Crown  octavo,  338  pages,  illustrated.  With  an 
Appendix  on  Antiseptic  Surgery,  etc.  Cloth,  $1.00  net.  In  Saunders' 
Question  Compends. 

Martin's   Essentials  of  Minor  Surgery,   Bandaging, 

and  Venereal   Diseases.       Second  Revised  Edition 

Essentials  of  Minor  Surgery,  Bandaging,  and  Venereal  Dis- 
eases. By  Edward  Martin,  A.  M.,  M.  D.,  Professor  of  Clinical  Sur- 
gery, University  of  Pennsylvania,  etc.  Crown  octavo,  166  pages,  with 
78  illustrations.     Cloth, ^i. 00  net.     In  Saunders'  Question  Compends. 


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